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1997

Characterization of Adaptive Changes in 5-HTIA Systems Induced by Repeated Injections of 5-HT Uptake Inhibitors

Qian Li Loyola University Chicago

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LOYOLA UNIVERSITY CHICAGO

CHARACTERIZATION OF ADAPTIVE CHANGES IN 5-HT1A RECEPTOR

SYSTEMS INDUCED BY REPEATED INJECTIONS OF 5-HT UPTAKE

INHIBITORS

A DISSERTATION SUBMITTED TO

THE FACULTY OF THE GRADUATE SCHOOL

IN CANDIDACY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY

AND EXPERIMENT AL THERAPEUTICS

BY

QIAN LI

CHICAGO, ILLINOIS

JANUARY, 1997 Copyright by Qian Li, 1996 All rights reserved.

ii ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Louis D. Van de Kar, for his remarkable guidance in the scientific process and experimental techniques during the entire duration that I worked in his laboratory. I am greatly indebted to him for helping me to develop the confidence necessary for my career. I am especially grateful to Dr. Van de Kar for the enormous amount of time he has spent with me and for his patience in helping me to improve my English.

I am grateful to my dissertation committee members, Drs. George Battaglia,

Thackery S. Gray, Israel Hanin, Mary D. Manteuffel and Nancy A. Muma for their suggestions and time spent on my dissertation project. I would especially like to thank

Dr. Battaglia and Dr. Muma for their tremendous technical support on my dissertation studies. In addition, I would like to thank Dr. Hanin for the instruction on how to survive not only in graduate school but also in the rest of my career.

I would also like to thank my colleagues Dr. Theresa Vera, Wilfred Pinto,

Kayoko Kunimoto, Francisca Garcia, Christopher Chambers, Gerry Newfry, Dayne

Okuhara, Alexsandra Vicentic, Joseph Yracheta, Dr. Andrew Levy and Dr. Peter

Rittenhouse for their generous and consistent help in all the experiments that present in this dissertation and for their wonderful friendship.

I would like thank all the faculty and staff of the Department of Pharmacology for their support and providing educational courses. I am grateful to the Graduate

iii School of Loyola University and to the Pharmaceutical Research and Manufacturers of

America Foundation Association for financial support.

I would like to thank Dr. Zheng-Tai Wang, the advisor for my masters degree,

for leading me to the scientific door and building up my interest in research. I appreciate his strict training in biochemistry and logical thinking which I have greatly benefited from while pursuing my doctoral degree, and will continue to benefit from

Throughout my career.

I am grateful to my parents, Shi-Ying Li and Jin Shen, for being an example of how to be a person and how to work, and for their constant support even when they were having very difficult times. Finally, I am deeply grateful to my husband

Liansheng and my son Chang, for their unending support and understanding. It would have been impossible for me to finish my studies without all the support from my family.

iv TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... iii

LIST OF ILLUSTRATIONS ...... x

LIST OF TABLES ...... xii

LIST OF ABBREVIATIONS ...... xiii

ABSTRACT .xv

Chapter

I. INTRODUCTION 1

II. REVIEW OF RELATED LITERATURE ...... 7

Serotonergic Systems ...... 7

General review of systems ...... 7

5-HT1A receptor systems ...... 10

Structure and function ...... 10

Signal transduction of 5-HT1A receptors ...... 14

5-HT IA agonists and antagonists ...... 17

5-HT1A receptor-mediated neuroendocrine responses ...... 19

5-HT1A receptor-mediated ACTH and corticosterone secretion ...... 20

5-HT1A receptor-mediated oxytocin secretion ...... 23

5-HT1A agonist-induced increase of secretion may not be mediated by 5-HT1A receptors ...... 25

Neuroendocrine challenge test . 27

5-HT1A Receptors and 5-HT Uptake Inhibitors 29

v 5-HT and affective disorders ...... 29

Effects of 5-HT uptake inhibitors on 5-HT1A receptor systems . . 32

Selective reuptake inhibitors (SSRI's) ...... 32

5-HT1A receptors in the therapeutic effects of SSRI's . . 35

The mechanism of desensitization of 5-HT1A receptors induced by chronic administration of 5-HT uptake inhibitors ...... 42

III. MATERIALS AND METHODS ...... 46

Animals ...... 46

Drugs and Reagents ...... 46

Biochemical Determinations 47

Radioimmunoassays . 47

Plasma ACTH radioimmunoassay ...... 47

Plasma corticosterone radioimmunoassay 48

Plasma oxytocin radioimmunoassay ...... 49

Radioligand binding assay . . . . 50

Homogenate binding assays for 5-HT1A receptors . 50

Autoradiographic analysis of 3H-8-0H-DPAT binding . . . 53

Immunoblot analysis of G proteins . . . . . 55

Membrane preparation ...... 55

Quantification of G proteins . . . . 56

Data analysis ...... 56

Characterization of antisera 57

Vl Statistics ...... 59

IV. LONG-TERM , BUT NOT , PRODUCES

A DESENSITIZATION OF HYPOTHALAMIC 5-HT1A RECEPTORS . 60

Summary ...... 60

Introduction ...... 61

Experimental Protocol ...... 63

Results ...... 64

Discussion ...... 74

V. TIME-COURSE OF FLUOXETINE-INDUCED DESENSITIZATION

OF HYPOTHALAMIC 5-HT1A RECEPTORS ...... 80

Summary ...... 80

Introduction ...... 82

Experimental Protocol ...... 85

Results ...... 86

Hormone responses to 8-0H-DPAT ...... 86

Autoradiographic analysis of 3H-8-0H-DPAT binding ...... 91

Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding . . . . . 98

G°' subunits in , midbrain and frontal cortex . . . . . 103

Discussion ...... 110

Hormone responses to 8-0H-DPAT ...... 110

Autoradiogram of 3H-8-0H-DPAT binding ...... 112

Autoradiographic analysis of Gpp(NH)p-induced inhibition of 3H- 8-0H-DPAT binding ...... 116

vii Changes in the levels of Gi and G0 proteins induced by repeated injections of fluoxetine ...... 117

VI. REPEATED INJECTIONS OF PRODUCE A

GRADUAL DESENSITIZATION OF 5-HT1A RECEPTORS IN THE HYPOTHALAMUS ...... 122

Summary ...... 122

Introduction ...... 125

Experimental Protocol ...... 128

Results ...... 129

Hormone responses to 8-0H-DPAT ...... 129

Autoradiographic analysis of 3H-8-0H-DPAT binding ...... 134

Levels of Gi and G0 proteins in the hypothalamus, midbrain and frontal cortex ...... 134

Discussion ...... 144

VII. GENERAL DISCUSSION ...... 150

The Neuroendocrine Challenge Test --- A Tool to Examine the Function

of Hypothalamic 5-HT1A Receptors ...... 150

Comparison of Desipramine with Fluoxetine in Neuroendocrine Challenge tests ...... 154

Comparison of the Effects of Fluoxetine with Paroxetine on

Hypothalamic 5-HT1A Receptors ...... 155

Possible Mechanism for the Desensitization of Hypothalamic 5-HT1A Receptors ...... 157

Significance of the Present Studies ...... 162

Limitation of the Present Studies ...... 165

Conclusions ...... 167

viii REFERENCES ...... 170

VITA ...... 202

lX LIST OF ILLUSTRATIONS

Figure Page

1. Characterization of antibodies against Gil, Gi2 , Gi3 and G0 proteins used in immunoblots...... 58

2. Effect of chronic exposure to fluoxetine or desipramine on ACTH responses to 8-0H-DPAT or ...... 67

3. Effect of chronic exposure to fluoxetine or desipramine on the corticosterone response to 8-0H-DPAT or ipsapirone ...... 68

4. Effect of a single injection of fluoxetine on ACTH and corticosterone responses to 8-0H-DPAT ...... 69

5. Effect of chronic exposure to fluoxetine or desipramine on the oxytocin response to 8-0H-DPAT or ipsapirone ...... 70

6. Effect of a single injection of fluoxetine on the oxytocin response to 8- 0H-DPAT ...... 71

7. Daily injections of fluoxetine inhibit the effect of 8-0H-DPAT on plasma ACTH ...... 88

8. Daily injections of fluoxetine inhibit the effect of 8-0H-DPAT on plasma corticosterone ...... 89

9. Daily injections of fluoxetine inhibit the effect of 8-0H-DPAT on plasma oxytocin ...... 90

10. Autoradiogram of 3H-8-0H-DPAT binding and Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding in the hypothalamus . . . . . 93

11. Autoradiogram of 3H-8-0H-DPAT binding and Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding in the midbrain and frontal cortex ...... 95

x 12. Degree of Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding varies between regions ...... 99

13. Example of immunoblots of G proteins in brain regions from rats that received daily injections of fluoxetine ...... 104

14. Fluoxetine reduces the levels of G proteins in the hypothalamus 105

15. Fluoxetine reduces the levels of G proteins in the mid brain. . . 107

16. Daily injections of paroxetine inhibit the effect of 8-0H-DPAT on plasma ACTH...... 131

17. Daily injections of paroxetine inhibit the effect of 8-0H-DPAT on plasma corticosterone...... 132

18. Daily injections of paroxetine inhibit the effect of 8-0H-DPAT on plasma oxytocin...... 133

19. Paroxetine reduces the levels of G proteins in the hypothalamus. 138

20. Paroxetine reduces the levels of G proteins in the midbrain...... 140

21. Paroxetine reduces the levels of G proteins in the frontal cortex...... 142

Xl LIST OF TABLES

Table Page

I. Pharmacologic parameters of 5-HT uptake inhibitors 34

II. Changes in Bmax of 5-HT1A receptors induced by chronic exposure to fluoxetine and desipramine ...... 72

III. Chronic fluoxetine or desipramine does not change the affinity of 5- HT1A receptors and 5-HT uptake sites ...... 73

IV. Repeated injections of fluoxetine do not alter the density of 5-HT1A receptors ...... 96

V. Repeated injections of fluoxetine do not alter the percent of - linked 5-HT1A receptors ...... 101

VI. Daily injections of fluoxetine do not alter levels of G-proteins in the frontal cortex ...... 109

VII. Repeated injections of paroxetine do not alter the density of 5-HT1A receptors at any brain region ...... 136

xii LIST OF ABBREVIATIONS

5-HIAA 5-Hydroxyindole acetic acid

5-HT serotonin

5-HTP 5-hydroxy-

5,7-DHT 5, 7-dihydroxytryptamine

8-0H-DPAT 8-hydroxy-2-(dipropy lamino )tetralin

ACTH adrenocorticotropin

ANOVA analysis of variance

CRF corticotropin-releasing factor

CRH corticotropin-releasing hormone cAMP cyclic adenosine monophosphate

DAG diacylglycerol

EEDQ N-ethoxycarbonyl-1,2-ethoxydihydroquinoline

GAIP G Alpha Interacting Protein

HPA axis hypothalamic-pituitary-adrenocortical axis

G protein guanine nucleotide-binding protein

GDP Guanosine diphosphate

GTP Guanosine triphosphate

IOD integrated optical density

xiii IP3 inositol triphosphate

MAO monoamine oxidase

PCA p-chloroamphetamine

PCPA p-chloropheny !alanine

PVN paraventricullar nucleus hypothalamus

RGS Regulators of G protein signaling

SSRI's Selective serotonin reuptake inhibitors

35 35 [ S]-GTP'YS S-guanosine-5 '-0-(thiotriphosphate)

xiv ABSTRACT

The purpose of the dissertation was to characterize the adaptive changes in 5-

HT 1A receptors induced by repeated injections of 5-HT uptake inhibitors. Hormones

released in response to 5-HT1A agonists were used as markers to determine the function

of hypothalamic 5-HT1A receptors. The density of 5-HT1A receptors, their coupling to

G proteins and the levels of Gi and G0 proteins were examined. Daily injections of 5-

HT uptake inhibitor fluoxetine for three weeks significantly reduced the ACTH,

corticosterone and oxytocin responses to the 5-HT1A agonists 8-0H-DPAT and ipsapirone. In contrast, daily injections of desipramine or acute injection of fluoxetine

did not alter the hormone responses to 5-HT1A agonists. Fluoxetine injections did not

reduce the affinity or density of 5-HT1A receptors in the hypothalamus. An examination of the time-course of the effect of fluoxetine and paroxetine revealed that both produce a gradual reduction in the ACTH, corticosterone and oxytocin responses to 8-

0H-DPAT. The reduction in hormone responses to 8-0H-DPAT appeared after three days and reached a maximum after seven to fourteen daily of injections. No change

was observed in the density of 5-HT1A receptors and in their G protein coupling in the hypothalamus or in other brain regions after daily injections of fluoxetine or paroxetine.

The time course of both drugs in reducing the hypothalamic level of Gi3 proteins was similar and parallel to the time course of the decrease in the hormone responses to 8-

xv OH-DPAT. This similarity suggests that the reduction in the level of Gi3 proteins may

be involved in the desensitization of hypothalamic 5-HT1A receptors. In conclusion, our results suggest that prolonged blockade of 5-HT uptake sites, but not of

uptake sites, produces a delayed and gradual desensitization of hypothalamic 5-HT1A receptors. This desensitization may be mediated by alterations in signal transduction

mechanisms of 5-HT1A receptors in the hypothalamus.

xvi CHAPTER I

INTRODUCTION

Selective serotonin (5-HT) reuptake inhibitors (SSRI's), such as fluoxetine

(Prozac®), (Luvox®), paroxetine (Paxil®), (Zoloft®) and are widely used to treat affective disorders, including depression, premenstrual disorder, obsessive compulsive disorder, bulimia/anorexia nervosa and anxiety (Advokat and Kutlesic, 1995; Wong et al.1995; Lam et al.1995; Tollefson et al.1994; Lecrubier, 1993; Sternlicht, 1993). Serotonin reuptake is the principal process responsible for terminating the action of 5-HT at post-synaptic receptors. By inhibiting the uptake of 5-HT into the nerve terminals, SSRI's act to increase the 5-HT concentration in the synaptic cleft. However, since there is a delay of about 14-21 days in clinical improvement after the initiation of SSRI administration, the therapeutic effects of these drugs may be mediated by adaptive changes in serotonergic neurotransmission, initiated by the inhibition of 5-HT uptake sites (Blier and de

Montigny, 1994; Montgomery, 1994; Briley and Moret, 1993; Richelson, 1991). It has

been suggested that dysfunction of 5-HT1A receptor systems may contribute to the etiology of affective disorders (Lesch et al.1992c; Lesch et al.1991b; Lesch, 1991).

Furthermore, it has been hypothesized that somatodendritic 5-HT1A receptors are involved in the delay of the onset of clinical improvement after treatment with SSRI's

(Gardier et al.1996; Briley and Moret, 1993). Therefore, studying the effect of 5-HT 2 uptake inhibitors on the 5-HT1Areceptor system can contribute to the understanding and improvement of the therapeutic effects of 5-HT uptake inhibitors.

5-HT IA receptors can be classified into somatodendritic 5-HT IA receptors

(autoreceptors) and postsynaptic 5-HT1A receptors. Somatodendritic 5-HT1A autoreceptors are located on serotonergic perikarya in the dorsal and median raphe nuclei in the midbrain (Le Poul et al.1995; Kreiss and Lucki, 1992). These 5-HT1A autoreceptors function as a negative feedback mechanism. Activation of 5-HT1A autoreceptors in the raphe decreases the firing rate of 5-HT neurons and, subsequently, reduces 5-HT release from their nerve terminals in the forebrain (Kreiss and Lucki,

1995; Kreiss and Lucki, 1992). Postsynaptic 5-HT1A receptors are widely distributed in the brain and are located on neurons that are innervated by serotonergic pathways

(Khawaja, 1995; Kung et al.1995; Wright et al.1995). The function of postsynaptic 5-

HT1A receptors is related to the function of target cells. For example, activation of 5-

HT1A receptors in the hypothalamus increases the secretion of ACTH, corticosterone and oxytocin (Fletcher et al.1996; Critchley et al.1994a; Van de Kar and Brownfield,

1993; Gilbert et al.1988a; Bagdy and Makara, 1994; Bagdy and Kalogeras, 1993).

Therefore, hormone responses to 5-HT1A agonists can be used as a peripheral marker of the functional status of hypothalamic 5-HT1A receptor systems. Somatodendritic 5-

HT1A receptors are more sensitive to 5-HT1A agonists than the postsynaptic 5-HT1A receptors. The doses of 5-HT1A agonists (such as 8-0H-DPAT, ipsapirone or ) that activate somato-dendritic autoreceptors to suppress the firing rate of dorsal raphe neurons are about 10-fold lower (Hjorth and Magnusson, 1988; Sprouse 3 and Aghajanian, 1988; Aghajanian et al.1990), than the doses that activate post-synaptic hypothalamic 5-HT1A receptors and increase the secretion of hormones such as ACTH

(Pan and Gilbert, 1992) or oxytocin (Bagdy and Kalogeras, 1993).

Several investigators have hypothesized that long-term exposure to 5-HT uptake inhibitors will first desensitize somatodendritic 5-HT1A autoreceptors, leading to increase release of 5-HT in the forebrain, and subsequently desensitize postsynaptic 5-

HT1A receptors (Le Poul et al.1995; Bel and Artigas, 1992; Blier and Bergeron, 1995;

Blier and de Montigny, 1994). Inhibition of 5-HT uptake sites results in an increase of concentration of 5-HT in the synaptic cleft, thereby activating 5-HT1A receptors.

Since 5-HT1A autoreceptors are more sensitive to the increase of 5-HT concentration than postsynaptic 5-HT1A receptors, the 5-HT1A autoreceptors are predominantly activated after administration of 5-HT uptake inhibitors. The activation of 5-HT1A autoreceptors will decrease the firing rate of 5-HT neurons and subsequently reduce the release of 5-HT from serotonergic nerve terminals (Arborelius et al.1995; Gartside et al.1995). Therefore, although forebrain 5-HT uptake sites are still blocked during this period, the concentration of 5-HT in forebrain synaptic clefts would not be increased.

Following repeated administration of SSRI's, somatodendritic 5-HT1A receptors are desensitized by consistent increase of 5-HT concentration in the presynaptic cleft of 5-

HT neurons. The desensitization of somatodendritic 5-HT1A autoreceptors would eventually free 5-HT neurons; firing would be resumed and 5-HT release would return to normal. Since forebrain 5-HT uptake sites are still inhibited by 5-HT uptake inhibitors, the concentration of 5-HT in the postsynaptic cleft would increase and induce 4 desensitization of postsynaptic 5-HT1A receptors, such as hypothalamic 5-HT1A receptors. If this hypothesis is correct, then repeated injections of fluoxetine would

produce a delayed and gradual reduction in the function of hypothalamic 5-HT1A receptors. Several investigators have provided evidence that 5-HT uptake inhibitors first produce inhibition of the firing rate of 5-HT neurons and decrease the release of

5-HT in the forebrain, followed by desensitization of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus as early as 3 days after administration of 5-HT uptake inhibitors (Le Poul et al.1995). However, very little is known regarding the

onset of desensitization of postsynaptic 5-HT1A receptors after repeated injections of 5-

HT uptake inhibitors.

5-HT1A receptors are coupled to G0 and/or Gi proteins (Raymond et al.1993;

Fargin et al.1991; Sprouse and Aghajanian, 1988; Emerit et al.1990). Somatodendritic

5-HT1A autoreceptors in the raphe nuclei are coupled to G0 proteins, which increase the

opening of K+ channels (Sprouse and Aghajanian, 1988). Activation of 5-HT1A receptors that are coupled to Gi proteins inhibits the activity of and consequently decrease the intracellular concentration of cAMP (cyclic adenosine monophosphate) (Varrault et al.1994). 5-HT lA receptors have high affinity for Gi

proteins with the rank order of affinity being Gi3 > Gil > Gi2 > G0 proteins (Raymond

et al.1993). Most of the studies having investigated the coupling of 5-HT1A receptors to G proteins, however, were conducted in cells in culture transfected with expressing

cloned 5-HT1A receptors and G proteins. Little is known about specific G proteins that

are involved in specific 5-HT1A receptor-mediated functions in vivo. For example, it 5 is still unclear which G proteins mediate 5-HT1A receptor-induced increases of ACTH and oxytocin secretion and 5-HT1A receptor-inducted hypothermia.

The purpose of the present project was to investigate adaptive changes in the hypothalamic 5-HT1A receptor systems induced by daily injections of 5-HT uptake inhibitors. The hypothesis was that daily injections of 5-HT uptake inhibitors produce a delayed and gradual desensitization of the hypothalamic 5-HT1A receptors. This desensitization may be mediated by alterations in the signal transduction of 5-HT1A receptors. Three specific aims were designed to address the hypothesis. First, to examine whether long-term fluoxetine and desipramine influence 5-HT1A receptor systems. Fluoxetine is a widely used SSRI, while desipramine is a norepinephrine uptake inhibitor and a tricyclic . Comparing the effect of fluoxetine with desipramine on 5-HT1A receptors allowed us to understand whether the effects of fluoxetine are unique to SSRI's. In addition, the effects of acute fluoxetine on the 5-

HT lA receptors were examined in order to determine whether the effects of fluoxetine are mediated by the long-term administration. Secondly, the time-course of the effects of fluoxetine on 5-HT1Areceptor systems was determined. If 5-HT1Aautoreceptors play a role in the delay of therapeutic effects of SSRI's, daily injections of 5-HT uptake inhibitors should produce a delayed, gradual alteration in postsynaptic 5-HT1Areceptors in the hypothalamus. Finally, I determined whether the effects of fluoxetine on the 5-

HT1A receptors are mediated by the blockade of 5-HT uptake sites by examining the time-course of effects of paroxetine on the hypothalamic 5-HT1A receptors and comparing these effects with those observed in the fluoxetine study. Paroxetine is 6 another 5-HT uptake inhibitor with a different chemical structure from fluoxetine. It has a higher affinity for 5-HT uptake sites, shorter half-life than fluoxetine, and no active metabolites (Nemeroff, 1993). Therefore, if the effects of paroxetine on the 5-

HT1A receptors are similar to those induced by fluoxetine, one can conclude that the alterations in the 5-HT1A receptors induced by daily injections of fluoxetine or paroxetine are mediated by the sustained blockade of 5-HT uptake sites. The following biochemical and functional parameters of 5-HT1A receptors examined to achieve these aims. 1) Hormone responses to 5-HT1A agonists were used as functional markers of 5-

HT1A receptors. 2) The density of 5-HT1A receptors and their coupling to G proteins in the rat brain were measured using homogenate or autoradiographic analysis of 3H-8-

, 0H-DPAT binding. 3) Gil, Gi2 Gi3 and G0 proteins, which are coupled to 5-HT1A receptors, were examined by immunoblots. CHAPTER II

REVIEW OF RELATED LITERATURE

Serotoner1:ic Systems

General review of serotonergic systems

Serotonergic pathways are important neurotransmitter systems in the central nervous system. Serotonin neurons, projecting their axons to the forebrain, are located in the dorsal and median raphe nuclei and in the ventrolateral B9 cell group in the brain stem. The distribution of serotonergic projections from the dorsal and median raphe are different. For example, the striatum mainly receives projections from the dorsal raphe, while the hippocampus receives projections from the median raphe (Bonvento et al.1992; Kreiss and Lucki, 1994). The serotonin neurons in both median and dorsal raphe nuclei project to the hypothalamus, cortex and amygdala (Van de Kar and

Lorens, 1979; Sawchenko et al.1983; Petrov et al.1992). On the other hand, 5-HT neurons in the raphe nuclei receive 5-HT input from 5-HT axon collaterals or dendro­ dendritic junctions, which is related to feedback regulation of 5-HT release (Wang and

Aghajanian, 1978; Wang and Aghajanian, 1977).

Serotonin neurons synthesize serotonin from tryptophan, an essential amino acid.

The key enzyme for the synthesis of 5-HT is tryptophan hydroxylase, which converts tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP is decarboxylated to 5-HT by

7 8 aromatic L-amino acid decarboxylase. Serotonin is then transported to serotonergic terminals through axons and stored in vesicles in the nerve terminals. When 5-HT neurons are activated, the firing rate of the neurons is increased, resulting in 5-HT release from nerve terminals to the synaptic cleft. In the synaptic cleft, 5-HT can bind to and stimulate 5-HT receptors, resulting in physiological responses. 5-HT, in the synaptic cleft, can also be taken back into the nerve terminal via 5-HT uptake sites (i.e.

5-HT transporters), which are located on the membrane of nerve terminals. In the nerve terminals, 5-HT can be restored in the vesicles or metabolized in the mitochondria to 5-HIAA (5-hydroxyindole acetic acid) by monoamine oxidase (MAO).

5-HT receptors are located on pre- or postsynaptic membranes. To date at least fifteen types of 5-HT receptors have been found by receptor-cloning studies (Hoyer et al.1994). The 5-HT receptors are classified into 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5 ,

5-HT6 and 5-HT7 receptors (Saxena, 1995; Hoyer et al.1994). Among them, 5-HT 1 receptors are subclassified to 5-HT1A, 5-HTrn, 5-HT10, 5-HTrn, and 5-HT1F receptors.

5-HT2 receptors are subclassified to 5-HT2A, 5-HT28 and 5-HT2c receptors. 5-HT2A

(previously 5-HT2) receptors and 5-HT2c (previously 5-HT1c) receptors have similar structures and few agonists or antagonists can distinguish between them. However, 5-

HT2c receptors have a higher affinity for 5-HT than 5-HT2A receptors. Also, 5-HT2c receptors are abundant in the choroid plexus. 5-HTrn and 5-HT10 receptors are autoreceptors. They are located in the nerve terminals and function as feedback regulators of 5-HT release. 5-HTrn receptors are found in rodent species, such as rats

and mice, while 5-HT10 receptors are located in non-rodent mammalian species, such 9 as guinea pig, pig, calf, rabbit, dog, monkey and . The distribution of 5-HTrn and 5-HTrn receptors in the brain is similar. Therefore, it is believed that 5-HTrn and

5-HT10 receptors play the same role in the different species. Except for 5-HT3 receptors which are cation channel-ligand gated receptors, all other 5-HT receptors are

G protein-coupled receptors. 5-HT1 receptors have a high affinity for 5-HT and are

coupled to Gi proteins, which inhibit adenylyl cyclase. Beside Gi proteins, 5-HT1A

receptors also couple to G0 proteins that increase opening of potassium channel and

2 inhibit the opening of Ca + channel. Except for 5-HT2c receptors, 5-HT2 receptors have relative lower affinity for 5-HT. Stimulation of 5-HT2 receptors will activate phospholipase C and, consequently increase the hydrolysis of inositol phospholipid to

2 inositol triphosphate (IP3) and diacylglycerol (DAG). DAG induces Ca + mobilization, which in tum activates protein kinase C, while IP3 activates calmodulin-dependent protein kinase. 5-HT4 , 5-HT6 and 5-HT7 receptors are positively linked to adenylyl cyclase. The signal transduction system of 5-HT5 is still unknown. Except for 5-HTrn and 5-HT 10 receptors that are located presynaptically, all other 5-HT receptors are located on postsynaptic membranes. Thus, their physiological functions are related to the function of the target cells. Physiological functions of 5-HTrn, 5-HT1p, 5-HT5 , 5-

HT6 and 5-HT7 receptors remain unknown. These 5-HT receptors were identified from cloned cDNA. Due to the lack of selective agonists and antagonists, few studies have been reported regarding their distribution and functions in the tissue of intact animals or .

In conclusion, serotonergic systems are involved in many regulatory processes 10 and play important roles in maintaining normal life. On the other hand, serotonergic systems are very complicated systems and very little is known about the systems. The

present dissertation is focused on the effects of SSRI's on 5-HT1A receptors. In the

following review, I will expand the discussion on the 5-HT1A receptor systems.

5-HT iA receptor systems

Structure and function

5-HT1A receptors belong to the G protein-coupled receptor family. Like other

receptors in this family, 5-HT1A receptors contain seven hydrophobic transmembrane domains, which are highly conserved with other G protein-coupled receptors, such as

B2 receptors (Raymond et al.1992). The connections of these seven membrane-spanning domains form three extracellular loops (ol-o3) and three

intracellular loops (il-i3). The N-terminal of 5-HT1A receptors faces the membrane

surface and the C-terminal is in the cytoplasm. The intracellular features of 5-HT1A receptors are similar to other cloned receptors which couple to Gi proteins, such as a long i3 loop and a short C terminal tail. These features are different from the receptors

coupled to Gs or Gq proteins, such as the B2 and 5-HT2A12c receptors, suggesting that the

i3 loop and C terminus may be involved in the coupling of 5-HT1A receptors to their

G proteins. Few studies on the function of 5-HT1A receptor domains in term of ligand binding and G protein coupling have been reported. Aspartate at positions 82 and 116

86 116 396 93 (Asp and Asp ), asparagine at position 396 (Asn ) and serine at position 393 (Ser3 ) may be important for ligand binding (Chanda et al.1993; Raymond et al.1992). Asp86 and Asp116 are located in transmembrane domain II and III respectively and Asn396 and 11

Ser393 are in transmembrane region VII. Except for these residues, the transmembrane

IV domain may also play a role in the binding of ligands to 5-HT1A receptors (Chanda et al.1993). It is interesting that about 70% of amino acids in transmembrane VI of 5-

HT1A receptors are identical to those in B receptors, suggesting that this domain may be related to the binding property of B antagonists to 5-HT1A receptors. Although 5-

HT1A receptors do not contain the sequence for phosphorylation by protein kinase A, at least 3 potential phosphorylation sites for protein kinase C have been found in the 5-

HT1A receptor. They are located in the i2 and i3 loops. These sequences may be the target for protein kinase C and induce a phosphorylation of 5-HT1A receptors, which would consequently produce a desensitization of 5-HT1A receptors (Nebigil et al.1995;

Raymond and Olsen, 1994; van Huizen et al.1993; Raymond, 1991). Furthermore, two cysteine residues in the C terminus of 5-HT1A receptors may be palmitoylated, which is one of the post-translational modifications that may modify the coupling of receptors to their G proteins (Raymond et al.1992).

5-HT1A receptors have been classified into somatodendritic and postsynaptic receptors (Hoyer and Boddeke, 1993; Blier et al.1993a; Blier et al.1993b). The somatodendritic 5-HT1A receptors (5-HT1A autoreceptors) are located on serotonin neurons in the dorsal and median raphe nuclei and receive serotonergic input from recurrent collaterals or from other raphe neurons (Wang and Aghajanian, 1978; Wang and Aghajanian, 1977; Sotelo et al.1990). The somatodendritic 5-HT1A receptors function as feedback regulators of 5-HT release from nerve terminals. When 5-HT neurons are activated, the firing rate of the neurons is increased, resulting in an 12 increase of 5-HT release from nerve terminals and recurrent collaterals. The increased concentration of 5-HT in the synaptic cleft of serotonin neurons activates somatodendritic 5-HT lA receptors. Consequently, the activation of somatodendritic 5-

HT 1A receptors inhibits the firing rate of 5-HT neurons and reduces 5-HT release from serotonergic nerve terminals (Hjorth and Sharp, 1991; Hjorth and Magnusson, 1988;

Bonvento et al.1992). Furthermore, activation of somatodendritic 5-HT1A receptors inhibits 5-HT synthesis in 5-HT neurons (Hjorth and Magnusson, 1988; Sharp et al.1993).

Postsynaptic 5-HT1A receptors are located on target cells in most forebrain

regions. The functions of postsynaptic 5-HT1A receptors are related to the target cells.

For example, 5-HT lA receptors in the ventromedial nucleus of the hypothalamus (VMN) may be involved in sexual behavior (Uphouse et al.1994a; Uphouse et al.1994b;

Mendelson and Gorzalka, 1986), and 5-HT1A receptors in the hippocampus may play a role in the feedback regulation of corticosteroid secretion (Burnet et al.1992; Lopez et al.1993; Zhong and Ciaranello, 1995; Chalmers et al.1993). As will be discussed

later, 5-HT1A receptors in the paraventricular nucleus of the hypothalamus may be involved in the regulation of ACTH, corticosterone and oxytocin secretion (Bagdy,

1995; Bagdy and Makara, 1994).

The density of 5-HT1A receptors varies among brain regions. The distribution

of 5-HT1A receptors in the brain has been studied using autoradiographic analysis of radioligand binding (Gozlan et al.1995; Khawaja, 1995; Le Poul et al.1995; Frankfurt et al.1994; Radja et al.1992; Dillon et al.1991; Hensler et al.1991; Marlier et al.1991; 13 Sijbesma et al.1991; Welner et al.1989; Palacios et al.1987) and immunocytochemistry

(Kia et al.1996). The hippocampus, septum and dorsal raphe contain the highest

density of 5-HT1A receptors, while the caudate putamen contains few 5-HT1A receptors.

Most of the studies on the autoradiography of 5-HT1A receptors have focused on the hippocampus and raphe nuclei. Very little is known regarding the distribution of 5-

HT lA receptors in the hypothalamus. A few investigators (Hensler et al .1991 ; Palacios et al.1987; Sijbesma et al.1991) have examined the density of 5-HT lA receptors in the hypothalamus, but only within a few nuclei, such as the ventromedial nucleus,

dorsomedial nucleus and lateral hypothalamus. So far, the distribution of 5-HT1A receptors in subdivisions of nuclei in the hypothalamus and amygdala has not been reported.

Several studies have compared the map of 5-HT1A receptors and the encoding

mRNA of 5-HT1A receptors (Miquel et al.1991; Chalmers and Watson, 1991;

Pompeiano et al.1992; Wright et al.1995; Raghupathi et al.1996). Generally, the

distribution of 5-HT1A receptors is consistent with their mRNA expression. A recent

study (Raghupathi et al.1996) showed that inactivation of 5-HT1A receptors by a high dose (10 mg/kg), but not by low doses (0.1and1 mg/kg) of EEDQ (N-ethoxycarbonyl-

1,2-ethoxydihydroquinoline) induced an increase of 5-HT1A receptor mRNA within 12 hours after injection. mRNA levels returned to normal, when the density of the receptors recovered. However, the magnitude of the increase of mRN A varied between

brain regions, whereas reduction of the density of 5-HT1A receptors was similar among

brain regions. Also, less than 503 inactivation of 5-HT1A receptors did not alter the 14 level of 5-HT1A receptor mRNA. These results suggest that changes in the 5-HT1A receptor mRNA may not be an exclusive mechanism of regulation of the density of 5-

HT1A receptors, especially when a modest change in the density of 5-HT1A receptors occurs.

Signal transduction of 5-HT iA receptors

5-HT1A receptors are G protein-coupled receptors. According to the model proposed by Lefkowitz et al. (1976), G protein-coupled receptors exist in high

(coupled) and low (uncoupled) affinity states with respect to agonists. Agonists only bind to receptors in high affinity states, while antagonists bind to both high and low affinity state receptors. The coupled receptor-G protein configuration only exists while

GDP (Guanosine diphosphate) is bound to the a-subunit of the G protein. When an agonist binds to receptors, the GDP on the G proteins coupled to the receptors will be exchanged with GTP (Guanosine triphosphate) due to changes in the conformation of the receptors. The replacement of GDP with GTP on the a-subunit of G proteins results in a dissociation of the G proteins from the receptors, which consequently switches the receptors from a high affinity-state to a low affinity-state. The dissociated

G proteins are then further dissociated into a and B'Y subunits. The a subunits activate a second messenger system, such as adenylyl cyclase and phospholipase C, which in turn activates the effectors and produces a physiological response. The a subunits are then inactivated by hydrolysis of the GTP into GDP via its intrinsic GTPase. The

GDP-bound a subunits will finally reassociate with the fJ'Y subunits and couple to receptors. 15 To date, about 20 G proteins have been identified and have been divided into 4

classes (Hamm and Gilchrist, 1996) based on their a subunit, Gs, Gi, Gq and G12 • Gs

proteins include Gs and G01f proteins. They activate adenylyl cyclase. Gi proteins

include Gil• Gi2 , Gi3 , G01 , G02 , Gz, Ggust and Gr proteins. Some of them (Gil• Gi2, Gi3

and Gz) inhibit adenylyl cyclase. G0 proteins increase the opening of potassium channels and decrease the opening of calcium (Ca2+) channels. Except for Gz proteins, all Gi proteins are pertussis toxin-sensitive, i.e pertussis toxin inactivates the Gi proteins

via ADP-ribosylation. Most of the Gq proteins (Gq, G11 , G14 and G15) stimulate phospholipase C and increase the intracellular concentration of inositol triphosphate and diacylglycerol.

Stimulation of hippocampal 5-HT1A receptors with 5-HT agonists inhibits forskolin-stimulated adenylyl cyclase activity (Yocca and Maayani, 1990; De Vivo and

Maayani, 1986). This inhibitory effect of 5-HT1A receptors can be blunted by pertussis

toxin, which selectively inactivates Gi and G0 proteins by ADP-ribosylation (Clarke et

al.1987). These results suggest that hippocampal 5-HT1A receptors are coupled to Gi

proteins. On the other hand, activation of 5-HT1A autoreceptors in the dorsal raphe does not inhibit forskolin-stimulated adenylyl cyclase activity (Yocca and Maayani,

1990; Clarke et al. 1990; Clarke et al. 1996), but does increase the opening of potassium channels, resulting in a hyperpolarization of 5-HT neurons (Sprouse and Aghajanian,

1988). This inhibitory effect of 5-HT1A agonists on cell firing and 5-HT release can also be blocked by pertussis toxin (Innis and Aghajanian, 1987; Innis et al.1988;

Romero et al.1994). These results suggest that 5-HT1A autoreceptors in the raphe are 16

coupled to G0 proteins. From these results, it is believed that 5-HT1A receptors are

coupled to Gi or G0 proteins. However, it is still unclear which particular Gi or G0 proteins are coupled to 5-HT1A receptors in specific cells, since at least three Gi and two

G0 proteins have been identified (Bimbaumer, 1993). Also, it cannot be ruled out that other G proteins, such as Gz or Gq proteins could couple to 5-HT1A receptors, since 5-

HT1A receptors are located on different cells and are involved in different functions.

However, it is difficult to study these questions in intact animals, since living cells are very complex and contain several different G proteins. Thus, most molecular biologic studies on the G protein-coupling of 5-HT1A receptors are performed using the expressional cloned 5-HT1Areceptors in a receptor-free cell line that does not naturally contain the 5-HT1A receptors.

Using co-expressed human 5-HT1A receptors and G proteins in Hela and CHO­

Kl cells, Raymond et al. (1993; 1994) found that 5-HT1A receptors have a high affinity

for Gi and G0 proteins. The rank order of the affinity for 5-HT1A receptors is Gi3 >

Gi1 > Gi2 > G0 proteins (Raymond et al.1993; Mulheron et al.1994; Bertin et al.1992).

Gs proteins do not seem to couple to 5-HT1A receptors (Raymond et al.1993; Bertin et al.1992; Butkerait et al.1995). These findings are consistent with biochemical and

A electrophysiological results that 5-HT1 receptors are coupled to Gi and/or G0 proteins.

However, in reconstituted cells, Gz protein, a pertussis toxin-insensitive member of the

Gi protein family, also strongly increases 3H-8-0H-DPAT binding (Butkerait et al.1995), suggesting that Gz proteins may be coupled to 5-HT1A receptors. In addition, in different receptor-negative cell lines, transfected 5-HT1A receptors express different 17 functions. For example, 5-HT1A receptors transfected into pituitary GH4 cells inhibit the activity of adenylyl cyclase, increasing the opening of potassium channels and closing calcium channels, but having no effect on phospholipase C. However, in Ltk

fibroblast cells (L cells), transfected 5-HT1A receptors not only inhibit adenylyl cyclase, but also stimulate phospholipase C and increase phosphoinositide turnover (Albert et

al.1996; Liu and Albert, 1991). Since all responses to 5-HT1A agonists in both cell

lines can be blocked by pertussis toxin, it is unlikely that 5-HT1A receptors in L cells

are coupled to Gq proteins. These results suggest that the functions of 5-HT1A receptors may vary in different cell environments. However, it should be noted that conditions in the cell lines are different from those in living cells. It is still a puzzle how G

proteins link to 5-HT1A receptors in vivo, since the natural cells are much more

complicated than cell lines. It is possible that the coupling of 5-HT1A receptors is not only dependent on their affinity for G proteins, but also dependent on the relative amount of each G protein in the cells (Raymond et al.1993).

5-HT tA agonists and antagonists

Several 5-HT1A agonists have been discovered such as 8-0H-DPAT, ipsapirone, , and buspirone (Hoyer et al.1994; Van Wijngaarden et al.1990).

Among them, 8-0H-DPAT has been considered to be the most selective 5-HT1A agonist

3 and is a full agonist for most 5-HT1A receptor-mediated responses. H-8-0H-DPAT is the most widely used radioactive ligand for examination of 5-HT1A receptors.

However, several studies have shown that 3H-8-0H-DPAT may bind to both high and

low affinity 5-HT1A receptors (Butkerait et al.1995). This could be due to the small 18 difference in affinity for 8-0H-DPAT between the high and low affinity-states of 5-

HT1A receptors (Kd=0.7nM vs. Kd=17nM) (Chamberlain et al.1993). Although ipsapirone and buspirone are partial 5-HT1A agonists, they have been frequently used in research since they can be administered to humans.

Few 5-HT1A antagonists are available. Most 5-HT1Aantagonists, such as NAN-

190 and WAY-100135, also behave as partial agonists (Escandon et al.1994; Cliffe et al.1993; Fletcher et al.1993; Gobert et al.1995; Greuel and Glaser, 1992; Claustre et al.1991b). is a 5-HT1A antagonist and can be used in humans. However, it also is a B . So far, the only silent 5-HT lA antagonist is WAY-

100635 (Forster et al.1995; Khawaja et al.1995; Routledge, 1995; Craven et al.1994).

Recently, several 5-HT1Aagonists and antagonists have been discovered, such as 8-0H­

PIPAT (5-HT1A agonist) (Zhuang et al.1993) and p-MPPI (5-HT1A antagonist) (Kung et al.1994). However, further studies on their pharmacologic profiles are still necessary.

5-HT1A agonists and antagonists bind to both somatodendritic and postsynaptic

5-HT1A receptors. However, some of them have a higher affinity for the somatodendritic 5-HT1A receptors than for postsynaptic 5-HT1A receptors (Matheson et al.1994). For example, the ED50 of (such as ipsapirone and buspirone) for inhibiting the firing rate of 5-HT neurons is much lower than the ED50 for stimulation

of postsynaptic 5-HT1A receptors (Matheson et al.1994). This may be due to a larger receptor reserve for 5-HT1A autoreceptors in the raphe than for postsynaptic 5-HT1A receptors in the forebrain (Cox et al.1993; Bohmaker et al.1992). 19

5-HT iA receptor-mediated neuroendocrine responses

Several 5-HT receptors are involved in the regulation of hormone secretion

(Tuomisto and Mannisto, 1985; Van de Kar et al.1995b; Van de Kar and Brownfield,

1993; Van de Kar, 1991 ; Yatham and Steiner, 1993; Murphy et al.1991 ; Fuller, 1992;

Fuller, 1990). For example, the secretion of ACTH, corticosterone and oxytocin can

be stimulated by 5-HT1A agonists (Van de Kar and Brownfield, 1993; Van de Kar,

1991). Stimulation of 5-HT2A;zc receptors will increase plasma levels of ACTH, corticosterone, renin, prolactin, oxytocin and vasopressin in a dose-dependent manner

(Rittenhouse et al.1994; Li et al.1993a; Rittenhouse et al.1993; Van de Kar and

Brownfield, 1993; Li et al.1992). Also, 5-HT18 and 5-HT3 receptors may be involved in the regulation of prolactin secretion (Levy et al.1995). 5-HT1A agonists do not stimulate renin and vasopressin secretion under normal conditions. In the present

review, the effect of 5-HT1A receptors on the regulation of hormone secretion will be discussed.

Most evidence has shown that hormonal regulatory effects are mediated by postsynaptic 5-HT lA receptors in the hypothalamus (Przegalinski et al.1989; Gilbert et

al.1988b). However, the signal transduction pathway of 5-HT1A receptor-mediated hormone secretion is still unknown. For example, which G proteins and second

messengers are involved in 5-HT1A receptor-mediated hormone secretion? How do

changes in second messengers trigger hormone release? Since 5-HT1A receptors may

be coupled to Gi or G0 proteins, it is possible that the hormone responses to 5-HT1A agonists are inversely related to the activity of adenylyl cyclase. 20

5-HT1A receptor-mediated ACTH and corticosterone secretion

The secretion of ACTH and corticosterone is regulated by the hypothalamic­

pituitary-adrenocortical axis (HPA axis). The paraventricular nucleus of the

hypothalamus (PVN) contains parvocellular cells that secrete corticotropin-releasing

hormone (CRH or CRF). When the parvocellular cells are activated, CRH is released

into the pituitary portal vessels and travels into the anterior lobe of the

through the superior hypophyseal artery. In the anterior pituitary (i.e

adenohypophysis), CRH stimulates adrenocorticotropin (ACTH) release from ACTH

containing cells (corticotrophs). ACTH then travels to the adrenal gland through the

circulation, where ACTH stimulates adrenal cortical cells resulting in release of

corticosterone in rats or cortisol in humans. The HPA axis includes both neurons and

endocrine cells and spans from the central nervous system to the peripheral adrenal

gland. It can be expected that the HPA axis will be regulated by both central and peripheral factors.

Several neurotransmitter systems mediate the regulation of ACTH and

corticosterone release. For example, activation of adrenergic a 1 receptors increases the

secretion of ACTH and corticosterone (Al-Damluji and Francis, 1993; Levy et al.1994;

Feldman et al.1995; Radant et al.1992). In addition, stimulation of D2

receptors and µ opiate receptors also increases ACTH and corticosterone release (Levy

et al.1994; Boesgaard et al.1990; Borowsky and Kuhn, 1992). It is worth noting that

D2 andµ receptors couple to Gi proteins, just as 5-HT1A receptors do. Therefore, it is

possible that these receptors share common G proteins, and hence, they can interact 21 with each other. Parvocellular cells (CRH cells) receive serotonergic input from dorsal and median raphe nuclei (Petrov et al.1992; Liposits et al.1987). Activation of several

5-HT receptors, such as 5-HT1A, 5-HTrn, 5-HT2Aizc and 5-HT3 receptors, increases

CRH, ACTH and corticosterone secretion (Fuller, 1995; Fuller, 1992; Fuller, 1990;

Fuller, 1981; Van de Kar and Brownfield, 1993; Van de Kar, 1991; Chaouloff, 1993).

Activation of 5-HT1A receptors by 5-HT1A agonists, such as 8-0H-DPAT, ipsapirone, buspirone and gepirone, increases plasma ACTH and corticosterone concentrations (Cowen et al.1990; Fuller, 1992; Gilbert et al.1988a; Koenig et

al.1988). The hormone responses to 5-HT1A agonists can be inhibited by 5-HT1A antagonists, such as pindolol, , NAN-190, UH-301, WAY-100135 and WAY-

100635 (Cowen et al.1990; Lejeune et al.1993; Pan and Gilbert, 1992; Critchley et al.1994b; Przegalinski et al.1989; Kelder and Ross, 1992; Vicentic et al.1996), but not by 5-HTrn, 5-HT2, 5-HT3 and adrenoceptor antagonists (Przegalinski et al.1989).

Several laboratories (Przegalinski et al.1989; Gilbert et al.1988b) and our preliminary data have shown that destruction of 5-HT neurons using 5,7-dihydroxytryptamine (5,7-

DHT) or depletion of 5-HT storage by p-chlorophenylalanine (PCPA) does not reduce

the ACTH and corticosterone responses to 5-HT1A agonists. These results suggest that

the ACTH and corticosterone responses to 5-HT1A agonists are exclusively mediated by

activation of postsynaptic 5-HT1A receptors. However, a study by Bluet Pajot et al.(1995) showed that infusing 8-0H-DPAT into the dorsal raphe nucleus slightly increases ACTH secretion. Also, in that study, repeated injections of PCPA decreased

ACTH responses to 8-0H-DPAT. These results suggest that presynaptic 5-HT1A 22 receptors may also be involved in the ACTH response to 8-0H-DPAT. However, no other data support these results so far and further confirmation is necessary.

Hypothalamic 5-HT1A receptors, especially those in the paraventricular nucleus, might be involved in the regulation of ACTH secretion (Bagdy and Makara, 1994;

Bagdy, 1994; Calogero et al.1989). Infusion of 8-0H-DPAT into the PVN significantly increases ACTH secretion (Bluet Pajot et al.1995). A lesion in the hypothalamic

paraventricular nucleus blocks the effect of the 5-HT1A agonist ipsapirone on plasma corticosterone concentration (Bagdy and Makara, 1994; Bagdy, 1994). Taken together,

these results suggest that the ACTH and corticosterone responses to 5-HT1A agonists are

mediated by postsynaptic 5-HT1A receptors in the hypothalamus.

A receptor-reserve has been demonstrated for the 5-HT1A receptor-mediated increase of plasma ACTH and corticosterone concentrations (Meller and Bohmaker,

1994; Pinto et al.1994). Irreversible inactivation of 5-HT IA receptors by EEDQ (N­ ethoxycarbonyl-1,2-ethoxydihydroquinoline) shifts the 8-0H-DPAT dose-response curves for plasma ACTH and corticosterone to the right. A low dose of EEDQ

(lmg/kg, sc) (Pinto et al.1994) reduced the density of 5-HT1A receptors by 53%, but did not change the hormone responses to 8-0H-DPAT. A high dose of EEDQ (6 mg/kg, sc) decreased both the potency and efficacy of ACTH and corticosterone responses to 8-0H-DPAT (Meller and Bohmaker, 1994). The fractional occupancy of

5-HT1A receptors required to reach 50% maximal response of ACTH or corticosterone is 25% or 5.5% respectively (Meller and Bohmaker, 1994). The effect of EEDQ on the hormone responses to 8-0H-DPAT can be prevented by pretreatment with the 5- 23

HT1A antagonist pindolol, suggesting that the effect of EEDQ on the ACTH and

corticosterone responses to 8-0H-DPAT is mediated by 5-HT1A receptors (Meller and

Bohmaker, 1994). The apparent receptor-reserve for 5-HT1A receptor-mediated ACTH and corticosterone may be due to the cascade of the HPA axis. When a hypothalamic

5-HT1A receptor is stimulated, the signals travel through the HPA axis from the hypothalamus, pituitary and adrenal cortical cells. The signals are amplified in each

step along the pathway, and thus, the response to the 5-HT1A agonist becomes amplified. This is consistent with data showing that the corticosterone response has more receptor reserve than the ACTH response.

5-HT1A receptor-mediated oxytocin secretion

Oxytocin is synthesized by magnocellular cells in the hypothalamic supraoptic and paraventricular nuclei. The oxytocin neurons send their axons to the posterior lobe of the pituitary gland where they secrete oxytocin into the inferior hypophyseal artery.

Oxytocin secretion can be regulated by stimulation of either oxytocin cell bodies in the hypothalamus or nerve terminals in the posterior pituitary. Several studies indicate that noradrenergic and serotonergic neurons are involved in the regulation of oxytocin secretion. Noradrenergic projections from neurons in the Al,A2 and A6 cell groups stimulate magnocellular cells in the paraventricular nucleus, while C2 noradrenergic

neurons inhibit the magnocellular cells (Saphier, 1993). Stimulation of a 1 receptors increases the secretion of oxytocin (Levy et al.1994; Saphier, 1993). In contrast with

ACTH, D2 and µ-receptors inhibit the secretion of oxytocin. Injection of the 5-HT releasers p-chloroamphetamine (PCA) and d- increase the concentration of 24 plasma oxytocin (Van de Kar et al.1995c; Saydoff et al.1991). The effect of 5-HT releasers can be inhibited by the 5-HT uptake inhibitor fluoxetine (Van de Kar et al.1995c). Neurons in the dorsal rap he nucleus innervate magnocellular cells in the paraventricular nucleus (Saphier, 1991). Furthermore, lesions in the paraventricular nucleus, but not in the supraoptic nucleus, by the cell-selective toxin ibotenic acid inhibits the oxytocin response to the 5-HT releaser p-chloroamphetamine (PCA) (Van de Kar et al. l 995c). These results suggest that serotonin neurons regulate oxytocin secretion by stimulating oxytocin neurons in the hypothalamic paraventricular nucleus.

Bagdy (1993) and our laboratory (Li et al.1993b) have found that activation of

5-HT1A receptors increases the secretion of oxytocin. 5-HT,A agonists, such as 8-0H­

DPAT and ipsapirone increase the concentration of plasma oxytocin in a dose dependent manner (Li et al.1993b; Bagdy and Kalogeras, 1993). Furthermore, the oxytocin response to 8-0H-DPAT can be inhibited by the 5-HT,A antagonist NAN-190 and

WAY-100635 (Vicentic et al.1996; Bagdy and Kalogeras, 1993). A lesion in the hypothalamic paraventricular nucleus decreases the effect of ipsapirone on the secretion of oxytocin (Bagdy, 1994), suggesting that neurons in the PVN are involved in the oxytocin response to 5-HT,A agonists. Unlike the 5-HT,A receptor-mediated ACTH

response, only a few studies have reported on the oxytocin responses to 5-HT1A agonists. The signal transduction mechanism of the 5-HT1A receptor-mediated regulation of oxytocin release is still not clear.

No data regarding receptor reserve for the oxytocin response to 5-HT IA agonists have been published so far. An observation by Pinto and Battaglia (unpublished) 25 indicates that less receptor reserve exists for the oxytocin response to 5-HT tA agonists than for ACTH. The oxytocin response to 8-0H-DPAT was examined in rats treated with different doses of EEDQ, to inactivate receptors in a graded manner. Although the Emax of oxytocin response to 8-0H-DPAT was not reduced with a low dose (1 mg/kg) of EEDQ, the 10 mg/kg dose of EEDQ completely blunted the oxytocin response to 8-0H-DPAT. This dose of EEDQ, however, did not completely block the

8-0H-DPAT-induced increase in plasma levels of ACTH.

A 5-HT1A receptor-mediated increase of oxytocin secretion occurs in both males and females. The physiologic significance of this response is still unknown. Recently, an interesting study (Bj6rkstrand et al.1996) found that an antagonist for oxytocin receptors inhibited the effects of 8-0H-DPAT on insulin, cholecystokinin and

somatostatin, suggesting that 5-HT1A receptor-mediated regulation of oxytocin secretion may be related to the regulation of other hormones via oxytocin receptors.

Unlike ACTH and corticosterone, oxytocin is released directly into the circulation from nerve terminals of oxytocin containing neurons in the hypothalamus.

Therefore, the magnitude of the oxytocin response to 5-HT1A receptors will more

directly reflect the function of 5-HT1A receptors in the hypothalamus than that of ACTH and corticosterone responses.

5-HT tA agonist-induced increase of prolactin secretion may not be mediated by 5- HT tA receptors

Prolactin is secreted from the anterior pituitary gland. The secretion of prolactin is regulated by the hypothalamus. It is believed that the hypothalamus produces a 26 releasing hormone to stimulate prolactin release. However, no prolactin releasing hormone has been found so far. Prolactin secretion is predominantly controlled by dopamine. Dopamine is released from the hypothalamus to the pituitary and inhibits secretion of prolactin. Therefore, inhibition of dopamine release or blockade of dopamine D2 receptors results in an increase in prolactin release.

Several 5-HT1A agonists, such as 8-0H-DPAT, ipsapirone and buspirone, stimulate prolactin secretion (Kellar et al.1992; Gartside et al.1990; Di Sciullo et al.1990; Aulakh et al.1988). Our preliminary data showed that destruction of 5-HT neurons using icv injection of 5, 7-DHT potentiated the prolactin response to 8-0H­

DPAT, suggesting that postsynaptic 5-HT1A receptors are involved in the 5-HT1A agonist-induced increase of prolactin release. In addition, injection of pertussis toxin,

which inactivates Gi and G0 proteins, or forskolin, an activator of adenylyl cyclase, inhibits the prolactin response to 8-0H-DPAT (Van de Kar et al.1995a). These results suggest that the prolactin response to 8-0H-DPAT is mediated by Gi proteins, which are negatively coupled to adenylyl cyclase. However, the time course and dose response of prolactin to 5-HT1A agonists are different from those of ACTH and corticosterone (Kellar et al.1992; Gartside et al.1990). The prolactin response to 5-

HT1A agonists can not be inhibited by the 5-HT1Aantagonists pindolol (Levy et al.1995;

Groenink et al.1995; Park and Cowen, 1995) or WAY -100635 (Vicentic et al.1996).

Furthermore, a surgical lesion in the hypothalamic PVN did not inhibit the prolactin response to ipsapirone (Bagdy and Makara, 1994). These results suggest that the prolactin response to 5-HT1A agonists may be not mediated by 5-HT1A receptors. 27 Neuroendocrine challenge test

In conclusion, 5-HT1A receptors are involved in the regulation of the secretion of ACTH, corticosterone and oxytocin. Therefore, the magnitude of the hormone

responses to 5-HT1A agonists can be used as a marker to examine the function of

hypothalamic 5-HT1A receptor systems.

As will be discussed below, dysfunction of serotonergic systems may play a role in affective disorders. Therapeutic effects of may be related to changes

in the function of 5-HT1A receptors. Since hypothalamic neurons are involved in mood

change, determination of the function of hypothalamic 5-HT1A receptors will help in the diagnosis and monitoring of the therapeutic effects of antidepressants (Van de Kar,

1989). Hormone responses to 5-HT agonists or releasers can be a useful peripheral tool to determine the function of central serotonergic systems. In fact, hormone responses to 5-HT agonists or releasers have been used clinically to assess the function of serotonergic systems in patients with affective disorders (Cowen et al.1994; Hollander et al.1991; Murphy et al.1991; Price et al.1991; Muller, 1990; Asnis et al.1988; Curtis and Glitz, 1988). For example, Lesch et al.(Lesch et al.1992c; Lesch, 1991; Lesch et al.1991b; Lesch et al.1991c; Lesch et al.1990a; Lesch et al.1990b; Lesch et al.1989) examined ACTH and cortisol responses to ipsapirone to assess changes in the function

of 5-HT1A receptors in depressed patients and the effect of antidepressants on 5-HT1A receptors. It is possible that changes in the function of 5-HT receptors occur prior to signs of clinical improvement during the treatment of affective disorders. Therefore, monitoring the function of 5-HT receptors using a neuroendocrine challenge test may 28 be a valuable tool to give physicians early information, such as whether the patient will respond to the treatment.

Although a neuroendocrine challenge test is a relatively reliable examination compared to other peripheral tests, its limitations should be noted. First, most agonists

(e.g. a 5-HT1A agonist) used as a challenge are not exclusively selective for one type

of 5-HT receptor (e.g 5-HT1A receptors). These drugs may also influence other neurotransmitter receptors (e.g. Dopamine D2 receptors). Therefore, the hormone

responses to the agonists may not only reflect the function of 5-HT receptors (5-HT1A

receptors), but also other receptors (D2 receptors). For example, buspirone is a 5-HT1A

agonist and an antagonist of dopamine D2 receptors. Since dopamine is a predominant inhibitory control for prolactin release, the prolactin response to buspirone will mainly

represent the function of D2 receptors. Secondly, since the pathways for 5-HT receptors regulating hormone release are still unknown and each hormone can be regulated by several different systems, it is possible that the steady-state of a hormone will be changed under different physical conditions, such as illness. In this case, the hormone challenge test may not represent the function of 5-HT receptors. To overcome these limitations, it is necessary to examine more than one hormone response. Since each hormone is regulated by different neurotransmitters, determination of several hormones will allow ruling out some of the side effects of agonists or abnormal hormone states. 29 5-HTlA Receptors and 5-HT Uptake Inhibitors

5-HT and affective disorders

Dysfunction of serotonergic systems is believed to be involved in several affective disorders, such as anxiety, depression, obsessive compulsive disorder, premenstrual syndrome and seasonal affective disorder (Sandyk, 1992; Shapira et al.1994; Eison, 1990; Mann et al.1990; Lesch et al.1992a). There is growing evidence regarding the involvement of abnormal serotonergic systems in schizophrenia and epilepsy (Rao and Moller, 1994; Holden, 1995; Statnick et al.1996). The evidence regarding the involvement of serotonergic systems in affective disorders includes the following: 1) Alterations of the concentration of 5-HT induce mood changes.

Delgado and his colleagues found that depletion of tryptophan, a precursor of 5-HT, by a tryptophan-free diet induced a relapse of depressive symptoms in remitted depressed patients (Delgado et al.1990). Also, tryptophan depletion worsened depressive symptoms in -free depressed patients one day after the test (Delgado et al.1994). In contrast, tryptophan depletion improved aggressive behavior one day after the test (Salomon et al.1994). These results indicate that abnormal serotonin levels may trigger mood changes, while the symptoms may be related to adaptive postsynaptic changes induced by the alteration of 5-HT (Delgado et al.1994). Consistent with the above results, a low 5-HT turnover (low 5-HIAA) in CSF has been found in impulsive violence offenders, which may be due to a genetic disorder (Virkkunen et al.1995). To determine whether the effect of tryptophan depletion on mood changes is due to a decrease of 5-HT release from nerve terminals, Bel and Artigas (1996) recently 30 examined the effect of tryptophan depletion on 5-HT concentration in rat frontal cortex using microdialysis. They found that tryptophan depletion significantly reduced 5-HT concentration in the frontal cortex of the rats that received fluvoxamine (a 5-HT uptake inhibitor) treatment. This result is consistent with the clinical observations. 2)

Changes in 5-HT receptors have been found in the brain of suicide victims. 5-HT

uptake sites were decreased and 5-HT1A receptors were increased in the ventrolateral prefrontal cortex of suicide victims (Arango et al.1995). Increased 5-HT2Aizc receptors in the cortex and amygdala and a low level of 5-HT1 receptors in the hippocampus have also been observed in suicides (Cheetham et al.1990; Hrdina et al.1993). However, the results from different studies were not consistent. This may be due to the variation in populations, sample sources and methods used between the studies. 3) Chronic exposure to antidepressants alters 5-HT transmission. The theories on the implication of dysfunction of serotonergic systems in the pathogenesis of affective disorders were originally based on the therapeutic effects of antidepressants (Shapira et al.1994). Most antidepressants alter 5-HT transmission, although the effective sites may vary between classes of antidepressants (Owens and Nemeroff, 1994). For example, tricyclic antidepressants tend to potentiate the function of postsynaptic 5-HT receptors, while SSRI's desensitize presynaptic 5-HT receptors (Blier et al.1987). 4).

5-HT uptake inhibitors are antidepressants As will be discussed below, 5-HT uptake inhibitors are widely used to treat affective disorders.

Taken together, these observations support the hypothesis that dysfunction of serotonergic systems may play a role in the pathophysiology of affective disorders. 31

However, since affective disorders are complicated processes, it is still unclear what the mechanisms of affective disorders are in terms of abnormalities of serotonergic systems.

5-HT1A receptors may play a role in the pathophysiology and therapeutics of affective disorders (Shapira et al.1994). A few studies observed abnormal functions of

5-HT1A receptors in patients with affective disorders. However, the observations were not consistent between studies. For example, hypothermic, ACTH and cortisol responses to ipsapirone were reduced in patients with unipolar depression, suggesting a decrease in the function of 5-HT1Areceptors in both pre- and postsynaptic sites (Lesch et al.1992c; Lesch, 1991; Lesch et al.1990b). However, the results reported by Cowen at al. (1994) reveal that hypothermia, but not ACTH and growth hormone responses to 5-HT1A agonists buspirone were attenuated in patients with major depression. This inconsistency may be due to the differences in population or type of the depression.

However, it should be noted that a dysfunction of 5-HT IA autoreceptors was consistently observed in both major and unipolar depressed patients. Although several mutated 5-

HT1A receptor genes have been found, none of them is correlated with depression

(Erdmann et al.1995; N akhai et al.1995; Xie et al.1995). In fact, more convincing evidence for existing dysfunction of 5-HT1A receptors in affective disorders comes from the involvement of 5-HT1A receptors in the therapy of affective disorders. Several 5-

HT1A agonists are anxiolytics and express antidepressive properties (De Vry, 1995;

Handley, 1995; Griebel, 1995). Also, some antidepressants, especially 5-HT uptake inhibitors (SSRI's), desensitize 5-HT1A receptors (Blier et al.1987; Lesch et al.1991c; 32 Lesch et al.1990a), as will be discussed later. Furthermore, it has been found that

administration of the 5-HT1A antagonist pindolol, combined with SSRI's, reduced the delay in the therapeutic effects of SSRI's (Artigas et al.1994; Blier and Bergeron,

1995).

Effects of 5-HT uptake inhibitors on 5-HT1A receptor systems

Selective serotonin reuptake inhibitors (SSRl's)

5-HT uptake inhibitors include fluoxetine (Prozac®), fluvoxamine (Luvox®), paroxetine (Paxil®), sertraline (Zoloft®) and citalopram. The 5-HT uptake inhibitors, especially fluoxetine, are widely used to treat depression, obsessive compulsive disorders, bulimia nervosa, panic disorder, obesity and premenstrual syndrome (Wong et al.1995; Goldstein et al.1995; Weltzin et al.1994; Advokat and Kutlesic, 1995;

Eriksson et al.1995; Steiner et al.1995; Woods et al.1993; Goldstein et al.1994a;

Goldstein et al.1994b; Lam et al.1995). A major advantage of the 5-HT uptake inhibitors is that they produce fewer side effects than the tricyclic antidepressants

(Wong et al.1995; Richelson, 1994).

5-HT uptake inhibitors do not share common chemical structures, but all have a high affinity for 5-HT uptake sites. They have a relatively low affinity for other monoamine transporters and receptors (Richelson, 1994; Wong et al.1995). Since the chemical structures of the 5-HT uptake inhibitors are different, their pharmacokinetic profiles are quite different as well. Among the 5-HT uptake inhibitors, fluoxetine has the lowest affinity, while paroxetine has the highest affinity for 5-HT uptake sites

(Richelson, 1994; Wong et al.1995). In humans, the half-life (t11i.) values for the 5-HT 33 uptake inhibitors range from 15 hours (paroxetine and fluvoxamine) to 1.9 days

(fluoxetine) after a single dose of administration (Table I). There is no active metabolite for paroxetine and fluvoxamine. However, the metabolite of fluoxetine, norfluoxetine, has a similar potency for 5-HT uptake sites and a much longer t112 (7 days) than fluoxetine. Although sertraline has an active metabolite, its activity is only

10% that of sertraline (Van Harten, 1993). It should be noted that the half-life values of the 5-HT uptake inhibitors are usually increased with chronic treatment (Van Harten,

1993; Goodnick, 1994). Very few studies have been reported regarding the pharmacokinetics of 5-HT uptake inhibitors in animals. Caccia et al. (1990) reported that the mean elimination half-life (t11z) of a single dose of fluoxetine in rats is about 5 hours (intravenous) or 7 hours (oral), while the t112 of norfluoxetine is about 15 hours.

These t112 values are much shorter than those in humans. However, the elimination half-life may not reflect the duration of effects of 5-HT uptake inhibitors in terms of ability to occupy 5-HT uptake sites. Scheffel et al. (1994) have shown that the t112 values of the inhibitory effect on 1251-RTI-55 binding (a in vivo radioactive tracer for

5-HT uptake sites) in the mouse brain are about 35, 7 and 6 hours for fluoxetine, sertraline and paroxetine, respectively.

The doses of 5-HT uptake inhibitors used in animals are determined as the doses that prevent 5-HT depletion by the 5-HT releasers PCA or fenfluramine, which enter nerve terminals through 5-HT uptake sites (Fuller et al.1978; Fuller and Wong, 1987). 34 TABLE I

PHARMACOLOGIC PARAMETERS OF 5-HT UPTAKE INHIBITORS

Fluoxetine Paroxetine Fluvoxamine Sertraline Citalopram Ki (nM)1 25 1.1 6.2 7.3 2.6 Ki ratio 20 320 180 190 1500 (NE: 5-HT)1 tmax (h)2 6-8 5 5 6-8 2-4 23 t 112 (human) '

parent compound 1.9 days 10-16 h 15 h 26 h 33 h

active metabolite 7 days NA NA 62-104 h t112 (rat)4,s

parent compound 5-7 h 3h

active metabolite 15 h

Dosage 20-80 20-30 100-300 50-200 3 6 (mg/day) '

NA: No active metabolite -: the data are unknown 1. Nemeroff, 1993; Van Harten, 1993 2. Van Harten, 1993 3. Goodnick, 1994 4. Caccia et al. 1990 5. Moret and Briley, 1990 6. Hollister and Claghorn, 1993 35

5-HT1A receptors in the therapeutic effects of SSRl's

The pharmacologic effects of 5-HT uptake inhibitors are blockade of 5-HT reuptake sites, resulting in an increase in the concentration of 5-HT in the synaptic cleft. However, like other antidepressants, the clinical improvement is usually observed two to three weeks after the onset of treatment with 5-HT uptake inhibitors. This delay of the clinical improvement suggests that the therapeutic effects of 5-HT uptake inhibitors might not be directly mediated by their acute pharmacologic effects, but are due to an adaptive change triggered by the sustained blockade of 5-HT uptake sites.

It has been hypothesized (Briley and Moret, 1993; Blier and de Montigny, 1994;

Gardier et al.1996) that 5-HT1A receptors are involved in the delayed onset of therapeutic effects of 5-HT uptake inhibitors. The blockade of 5-HT uptake sites by

5-HT uptake inhibitors leads to an increase in the concentration of 5-HT in the synaptic cleft both in the raphe and in postsynaptic brain areas (Fuller, 1994; Rutter and

Auerbach, 1993). Since somatodendritic 5-HT1A autoreceptors (in the raphe) are more sensitive to the alteration of 5-HT concentration in the synaptic cleft than postsynaptic

5-HT1A receptors (Blier et al.1993a; Hoyer and Boddeke, 1993), the increase in 5-HT

concentration in the synaptic cleft will first stimulate somatodendritic 5-HT1A

autoreceptors. The activation of 5-HT1A autoreceptors results in an inhibition in the firing rate of 5-HT neurons of the raphe nuclei and consequently, reduces 5-HT release from nerve terminals in the forebrain. Thus, initially, the net 5-HT concentration in the synaptic cleft of forebrain may not be increased. The decrease in 5-HT release may

even produce an initial supersensitivity of postsynaptic 5-HT1A receptors (Ceci et 36 al.1993). With repeated administration of 5-HT uptake inhibitors, it is hypothesized

that the over-stimulation of somatodendritic 5-HT1A autoreceptors by the high

concentration of 5-HT will produce a desensitization of somatodendritic 5-HT1A

autoreceptors in the raphe. The desensitization of somatodendritic 5-HT1A autoreceptors reduces the responses of serotonin neurons to 5-HT. Consequently, the 5-HT neurons will be liberated from the inhibition of firing activity and 5-HT release from 5-HT nerve terminals will return to normal or even be increased. Therefore, the concentration of 5-HT in the postsynaptic clefts will be increased, resulting in a

desensitization of postsynaptic 5-HT1A receptor systems. The desensitization of

postsynaptic 5-HT1A receptors may be implicated in the therapeutic effects of 5-HT uptake inhibitors.

Evidence to support this hypothesis will be discussed below. Most of the

evidence is focused on somatodendritic 5-HT1A receptors and very few studies have

investigated the adaptive changes in postsynaptic 5-HT1A receptors.

1) Inhibition of 5-HT neurons induced by acute injection of 5-HT uptake inhibitors is

mediated by somatodendritic 5-HT1A autoreceptors. Acute administration of 5-HT uptake inhibitors decreases the firing rate of 5-HT neurons in the dorsal and median rap he nuclei (Rigdon and Wang, 1991 ; Rutter and Auerbach, 1993; Chaput et al .1988;

Haj6s et al.1995). This effect can be blocked by the 5-HT1A antagonists pindolol, (S)­

UH-301, WAY-100135, spiperone and WAY-100635 (Arborelius et al.1995; Haj6s et al.1995; Gartside et al.1995; Huang and Harlan, 1994), suggesting that 5-HT uptake

inhibitor-induced reduction in the firing rate of 5-HT neurons is mediated by 5-HT1A 37 autoreceptors. Consistent with the electrophysiological results, it is limited that the influence of acute administration of 5-HT uptake inhibitors on extracellular concentration of 5-HT in the forebrain, which was measured by microdialysis.

Depending on the doses of 5-HT uptake inhibitors used, the extracellular 5-HT concentration in forebrain regions can be slightly increased (high doses of SSRI's) or not changed by systemic injections of 5-HT uptake inhibitors (Fuller, 1994; Gartside et al.1995; Gardier et al.1996; Rutter et al.1995; Rutter and Auerbach, 1993; Invernizzi et al.1992). Systemic injections of fluoxetine, paroxetine or sertraline (10 mg/kg, ip) to rats that received a local perfusion of fluoxetine into the diencephalon to block 5-HT uptake in the nerve terminals, significantly decreased the extracellular concentration of

5-HT in the diencephalon (Rutter et al.1995). This decrease of 5-HT release could be

reversed by systemic injections of 5-HT1A antagonists, such as WAY-100135 or

spiperone. Invernizzi et al. (1996) also observed that systemic injection of the 5-HT1A antagonist WAY-100635 potentiated the increase of extracellular 5-HT concentration in the frontal cortex induced by an acute injection of fluoxetine. Similar results have also been observed in the hippocampus by Hjorth et al. (1994a; 1995). Also, the increase of 5-HT concentration in the diencephalon induced by local infusion of fluoxetine was twice as high as that induced by systemic injection of fluoxetine (Rutter and Auerbach, 1993). Together, these data suggest that acute injections of 5-HT uptake inhibitors indirectly activate 5-HT1A autoreceptors on the 5-HT neurons, resulting in an inhibition of activity of 5-HT neurons and a decrease in 5-HT release. Furthermore, the increase in 5-HT concentration in forebrain regions, induced by 5-HT uptake 38 inhibitors, is much less than that in the raphe nucleus (Gartside et al.1995; Gardier et

al.1996; Rutter et al.1995). These results suggest that the stimulation of 5-HT1A autoreceptors induced by acute injection of 5-HT uptake inhibitors may not be only due

to the higher sensitivity of 5-HT1A autoreceptors compared with postsynaptic 5-HT1A receptors, but also due to a higher 5-HT concentration in the raphe regions than that

in the forebrain regions. However, it is unknown why activation of 5-HT1A autoreceptors does not inhibit the release of 5-HT in the raphe nuclei.

2) Long-term treatment with 5-HT uptake inhibitors desensitizes 5-HT1A autoreceptors in the raphe nuclei. Several studies have demonstrated that chronic treatment with 5-

HT uptake inhibitors reduces the ability of subsequent injections of 5-HT uptake inhibitors to decrease 5-HT neuronal activity. Blier et al. (1987; 1988) showed that the firing rate of 5-HT neurons was significantly decreased after 2 days of injections with the 5-HT uptake inhibitors , or citalopram. However, the firing rate of neurons was partially restored after 7 days and returned to normal after 14 days of injections of indalpine, zimelidine or citalopram. Repeated injections of fluoxetine, paroxetine or , a novel 5-HT uptake inhibitor, significantly shifted to the right the dose-response curve of 8-0H-DPAT- or ipsapirone-induced inhibition of the firing rate of 5-HT neurons in brain stem slices (Le Poul et al .1995; J olas et al .1994).

This reduction of the effect of 8-0H-DPAT on the firing of 5-HT neurons occurred after 3 days of repeated injections (Le Poul et al.1995). These results suggest that

repeated injections of 5-HT uptake inhibitors desensitize 5-HT1A autoreceptors in serotonin neurons. 39 Unlike the electrophysiological data, results from microdialysis studies to determine the effects of repeated injections of 5-HT uptake inhibitors on 5-HT release have not been as consistent (Gardier et al.1996). Bel and Artigas (1993) reported that repeated injections of fluvoxamine increased basal level of 5-HT in the frontal cortex, but not in the raphe region. The increase in the basal level of 5-HT induced by chronic fluvoxamine (2 weeks) was higher than acute fluvoxamine-induced increase of 5-HT concentration in the frontal cortex. Also, extracellular 5-HT levels in the frontal cortex of chronic fluvoxamine-treated rats were not further increased by a challenge with fluvoxamine. Several other investigators (Rutter et al.1994; Kreiss and Lucki, 1995;

Invernizzi et al.1996; Invernizzi et al.1994) found that repeated injections of fluoxetine or citalopram increased baseline concentration of 5-HT in several forebrain regions, such as frontal cortex, striatum, hippocampus and diencephalon. When a single dose of a 5-HT uptake inhibitor was used as a challenge, the extracellular concentration of

5-HT in the forebrain increased to a much higher degree in the rats that received repeated injections of 5-HT inhibitors than in the control rats. Furthermore, repeated injections of 5-HT uptake inhibitors, such as fluoxetine, abolished 8-0H-DPAT-induced inhibition of 5-HT release (lnvernizzi et al.1996). Taken together, these results suggest that repeated administration of 5-HT uptake inhibitors leads to a desensitization of 5-

HT1A autoreceptors on 5-HT neurons. This desensitization of 5-HT1A autoreceptors liberates serotonin neurons from the inhibition of firing activity and consequently, increases the concentration of 5-HT in the postsynaptic cleft. However, several studies do not support this hypothesis. Hjorth and Auerbach (1994b) reported that chronic 40 citalopram did not reduce the inhibitory effect of 8-0H-DPAT on 5-HT release in the frontal cortex and dorsal hippocampus. Also, an increased baseline of hypothalamic

5-HT in rats chronically treated with citalopram was only observed during administration, but not after a 24 hours washout (Moret and Briley, 1996). Although

Invernizzi et al. (1995) observed a desensitization of 5-HT1A autoreceptors induced by chronic fluoxetine, they did not find that chronic citalopram produced desensitization of 5-HT1A autoreceptors. These inconsistent results could be due to the different experimental procedures, different 5-HT uptake inhibitors used or different brain regions examined.

3) 5-HT1A antagonists shorten the delay of clinical improvement in the 5-HT uptake inhibitor therapy. If the hypothesis is correct that activation of 5-HT1A autoreceptors plays a role in the delay of the onset of clinical improvement after administration of 5-

HT uptake inhibitors, then, blocking the 5-HT1Aautoreceptors with 5-HT1A antagonists while administering 5-HT uptake inhibitors should speed the therapeutic effects of 5-HT uptake inhibitors. Artigas (1994; 1995) was the first to give depressed patients the 5-

HT1A antagonist pindolol with 5-HT reuptake inhibitors. Two-thirds of the patients remitted completely within 1 week. Some of them had taken 5-HT uptake inhibitors for at least 6 weeks with no full antidepressant responses before pindolol was added.

A similar result was also observed by Blier et al (1995). Nine patients with unipolar depression and 19 patients with resistant unipolar depression were treated with 5-HT uptake inhibitors and pindolol. Most of them achieved clinical improvement within 1 week. All patients, Except one, remitted within 14 days. Furthermore, Koran et al. 41 (1996) recently reported that pindolol also short the delay of the therapeutic effect of

5-HT reuptake inhibitors on obsessive compulsive disorder. All of these results support

the hypothesis that 5-HT1A autoreceptors are involved in the delay of clinical improvement during treatment with 5-HT uptake inhibitors. However, it should be noted that pindolol is also a B- antagonist. It still cannot be ruled out that the effect of pindolol in accelerating the therapeutic effect of 5-HT uptake inhibitors is due to its B antagonist effect. There were some patients that did not gain benefit from the combined pindolol and 5-HT uptake inhibitor treatment. All data, so far, have come from clinical trials and the number of cases is still limited. Therefore, further studies are necessary.

4) The effects of chronic 5-HT uptake inhibitors on postsynaptic 5-HT1A receptors. Data

regarding the effect of 5-HT uptake inhibitors on the function of postsynaptic 5-HT1A receptors are limited. Lesch et al.(199lc) reported that long-term treatment with

fluoxetine reduced the ACTH response to the 5-HT1A agonist ipsapirone in patients with obsessive-compulsive disorder. Hensler et al. (1991) showed that repeated injections of sertraline or citalopram attenuated the hypothermic response to 8-0H-DPAT, which

in rats is mediated by postsynaptic 5-HT1A receptors. These results suggest that chronic

injections of 5-HT uptake inhibitors desensitize postsynaptic 5-HT1A receptors.

In conclusion, evidence supports the hypothesis that the therapeutic effects of

SSRI's may be mediated by desensitization of 5-HT1A receptors. However, most studies

focus on the presynaptic 5-HT1A autoreceptors. Less is known about the effect of

chronic administration of SSRI's on postsynaptic 5-HT1A receptors. 42 The mechanism of desensitization of 5-HTlA receptors induced by chronic administration of 5-HT uptake inhibitors

The desensitization of 5-HT1A receptors is defined as a reduction in the function

of 5-HT1A receptor systems. It is still unknown what changes in the components of 5-

A A HT1 receptor systems contribute to the desensitization. 5-HT1 receptor systems

include the 5-HT1A receptors, G proteins that are coupled to 5-HT1A receptors, second messengers and effectors that couple to the second messenger and determine the

function of 5-HT1A receptors in the cells. Most studies have investigated the effects of

chronic 5-HT uptake inhibitors on the 5-HT1A receptors. Recently, some studies on the effects of 5-HT uptake inhibitors on G proteins, second messengers and the regulation of 5-HT receptors or G proteins have been reported (Newman et al.1992; Jolas et al.1994; Lesch and Manji, 1992; Lesch et al.1992b).

Several studies have shown that repeated injections of 5-HT uptake inhibitors do

not change the density of 5-HT1A receptors (either pre- or postsynaptic) (Le Poul et al.1995; Hensler et al.1991; Jolas et al.1994). Le Poul et al. (1995) reported that

neither 5-HT1A agonist binding (3H-8-0H-DPAT) nor 5-HT1A antagonist binding (3H-

WA Y-100635) was altered by repeated injections of fluoxetine or paroxetine. These

results indicate that the total number of 5-HT1A receptors and high affinity state 5-HT1A receptors are not changed by repeated injections of 5-HT uptake inhibitors. Hensler et al. (1991) found that although repeated injections of sertraline or citalopram attenuated the hypothermic response to 8-0H-DPAT, there were no changes in the density of 5-

3 HT1A receptors as measured by autoradiographic analysis of H-8-0H-DPAT binding.

In contrast, Welnet et al. (1989) reported that repeated injections of fluoxetine for 21 43 days decreased the density of 5-HT1A receptors in the dorsal raphe but not in the hippocampus. Also, Klimek et al. (1994) reported that citalopram, but not fluoxetine,

enhances the density of 5-HT1A receptors in the hippocampus.

Since repeated injections of 5-HT uptake inhibitors may not change the density

of 5-HT1A receptors or the changes only occur in specific nuclei, it is possible that the

desensitization of 5-HT1A receptors induced by 5-HT uptake inhibitors is mediated by

alterations of the signal transduction systems of 5-HT1A receptors. Newman, et al.

(1992) have found that chronic exposure to fluoxetine reduces 5-HT-induced inhibition of forskolin-stimulated adenylyl cyclase in rat hippocampus. In contrast with the observation, repeated injections of cericlamine (a new 5-HT uptake inhibitor) for 14 days did not reduce the 8-0H-DPAT-induced inhibition of forskolin-stimulated adenylyl cyclase, although the forskolin-induced activity of adenylyl cyclase was increased in the chronic cericlamine treated rats (Jolas et al.1994). No study has examined changes in

the coupling of 5-HT1A receptors to their G proteins, although Le Poul et al. (1995)

indicated that the ratio of 5-HT1A agonist and antagonist binding is not altered by repeated injections of 5-HT uptake inhibitors. Their data suggest that repeated injections

of 5-HT uptake inhibitors do not alter the coupling of 5-HT1A receptors to their G proteins. No data regarding changes in the levels of G proteins induced by repeated injections of 5-HT uptake inhibitors have been reported, although some investigators have studied the effects of tricyclic antidepressants on the levels of G proteins in several brain regions (Lesch et al.1991a; Lesch and Manji, 1992) and in glial cells (Chen and

Rasenick, 1995b). Lesch et al. (1992b; 1992) examined the changes in Gs, Gil• Gi2, G0 , 44

Gq and G 12 rnRN A expression after repeated administration of a low dose of fluoxetine for 21 days. Their results indicate that fluoxetine increases G12 rnRNA in the frontal

cortex, Gq rnRNA in the neostriatum and Gi2 and G0 rnRNA in the midbrain. No change in the G protein rnRNA was detected in the hypothalamus and hippocampus.

These results suggest that chronic administration of 5-HT uptake inhibitors may change the level of G proteins. All together, these results suggest that the desensitization of

5-HT1A receptors, induced by repeated injections of 5-HT uptake inhibitors, may not

be due to changes in the density of 5-HT1A receptors and their coupling to G proteins

in the steady state. It is possible that the desensitization of 5-HT1A receptors is mediated by alterations in the components of the signal transduction system, such as the level of G proteins and activity of adenylyl cyclase, resulting in dynamic changes in the

signal transduction system of 5-HT1A receptors in the neurons. Furthermore, the

regulation of 5-HT1A receptors and/or G proteins, for example by phosphorylation or palmitoylation (Wedegaertner et al.1995; Ross, 1995; Milligan et al.1995), could play

a role in producing the desensitization of 5-HT1A receptors. Several studies have

demonstrated that phosphorylation of 5-HT1A receptors induces their desensitization

(Nebigil et al.1995; Raymond and Olsen, 1994; van Huizen et al.1993; Raymond,

1991). Some of these studies have shown that the phosphorylation of 5-HT1A receptors may be mediated by protein kinase A and/or protein kinase C (Raymond and Olsen,

1994; Raymond, 1991). Others suggest that the phosphorylation of 5-HT1A receptors may be mediated by other mechanisms but not by protein kinase C (Nebigil et al.1995; van Huizen et al.1993; Claustre et al.1991a). Mann et al. (1995) reported that repeated 45 injections of fluoxetine (5 mg/kg) for 21 days significantly decreased the activity of protein kinase C in the cortex and hippocampus. Therefore, it is unlikely that the

desensitization of 5-HT1A receptors, induced by repeated injections of 5-HT uptake inhibitors, is due to the phosphorylation by protein kinase C. So far, no study has been

reported regarding the effects of 5-HT uptake inhibitors on the palmitoylation of 5-HT1A receptors, although there are some studies on palmitoylation of G proteins (Milligan et al.1995; Ross, 1995; Wedegaertner et al.1995; Degtyarev et al.1994). Taken together,

the mechanism of desensitization of 5-HT1A receptors, induced by chronic 5-HT uptake inhibitors, is still unclear. Specifically, no studies have reported on the time-course of

the effect of 5-HT uptake inhibitors on the 5-HT1A receptor system, including changes

in the 5-HT1A receptors, their signal transduction system, and 5-HT1A receptor-mediated physiological functions. CHAPTER III

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (225-275 g) were purchased from Harlan Sprague

Dawley Inc. (Indianapolis, IN). The rats were housed two per cage in a lighting (12 hr light/dark; lights on at 7 A.M), humidity and temperature-controlled room. Food and water were available ad lib. All procedures were conducted in accordance with the

NIH Guide for the Care and Use of Laboratory Animals as approved by the Loyola

University Institutional Animal Care and Use Committee.

Drugs and Reagents

Fluoxetine was a gift from Eli Lilly and Company (lndianapolis,IN).

Desipramine was purchased from Sigma Chemical Co (St. Louis, MO). Paroxetine was a gift from Smith-Kline Beecham. 8-0H-DPAT was purchased from Research

Biochemicals Inc (Natick, MA). Ipsapirone was a gift from Miles Laboratories, 'Inc.

(West Haven, Connecticut). All the drugs were dissolved in saline and injected in a volume of 2 ml/kg for antidepressants and 1 ml/kg for 8-0H-DPAT and ipsapirone.

ACTH antiserum was purchased from lgG Corp (Nashville, TN). ACTH (1-39) standards were obtained from Calbiochem (San Diego CA). Bovine serum albumin and aprotinin were purchased from Sigma Chemical Co. (St. Louis, MO). Normal rabbit

46 47 serum and goat anti-rabbit-')'-globulin were purchased from CalBiochem (San Diego,

CA). 1251-ACTH was obtained from INCSTAR (Stillwater, MN). Corticosterone antiserum was purchased from ICN Biochemicals (Irvine, CA). Ultima Gold scintillation fluid was purchased from Packard Instrument Co., Inc. (Downers Grove,

IL). acetone (Spectranalyzed A-19) and petroleum ether were obtained from Fisher.

3H-corticosterone, 1251-oxytocin and 3H-8-0H-DP AT were purchased from DuPont-NEN

(Boston, MA) at a specific activity of 129 Ci/mmol for the fluoxetine study and 135

Ci/mmol for the paroxetine study. Gpp(NH)p was purchased from Sigma Chemical

Co. (St. Louis, Mo). Recombinant a-subunits of G protein immunoblot standards were

purchased from Calbiochem (San Diego, CA). Anti Gi112 (AS/7) and anti G0 (GC/2) sera were purchased from Du-Pont NEN. Anti Gi3 serum was purchased from Upstate

Biotechnology Inc. (Lake Placid, NY). Rabbit peroxidase-antiperoxidase was purchased from Organon Teknika Co (Durham, NC). The chemiluminescence substrate solution

LumiGlo was obtained from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg,

MD). NP-40 (Nonidet P-40 or lgepal CA-630) was purchased from Sigma Chemical

Co. (St. Louis, Mo).

Biochemical Determinations

Radioimmunoassays

Plasma ACTH radioimmunoassay

ACTH radioimmunoassay was performed in an un-extracted plasma samples using an ACTH antiserum. The sequence recognition of the antiserum is the 5-18 segment of ACTH. This antiserum does not significantly recognize a-MSH, /j-MSH, 48

,6-endorphin, ,6-lipotropin, ACTH 11-24, or ACTH 1-16-amide. ACTH (1-39) was used as standards. In this double antibody radioimmunoassay, samples and standards were incubated with the ACTH antibody (final dilution 1:30,000, 223 total binding) at 4 °c for 24 hr in a 0. 01 M phosphosaline buffer (PBS, pH 7. 6) containing 1 % bovine serum albumin, 0.025 M EDTA, 0.53 normal rabbit serum and 25 KIU/ml aprotinin.

1251-ACTH (2000 cpm) was added and incubated for 24 hr at 4°C in a total volume of

0.3 ml. The second antibody (goat anti-rabbit-)'-globulin) was added at a final dilution of 1 :50 and incubated overnight at 4°C. After 1.5 ml of cold PBS was added, the tubes were then centrifuged at 15,000 x g 4°C for 40 min. The radioactivity of the pellet was counted for 5 min by a Micromedic 4/200 plus 'Y counter and analyzed from a standard curve using a RIA-AID computer program (Robert Maciel Associates, Arlington, MA).

The sensitivity of the assay is 0.25 pg/tube and the intra- and inter-assay variations are

4.23 and 14.6% respectively.

Plasma corticosterone radioimmunoassay

Corticosterone radioimmunoassay was performed on un-extracted plasma samples in which corticosterone-binding proteins had been denatured by boiling, as previously described (Van de Kar et al.1985). The assay is based on procedures and antiserum

(final dilution 1:11,200, 463 total binding) from ICN Biochemicals (Irvine, CA).

Briefly, plasma (2 or 5 µl) and standard (0-5 ng/tube) were incubated with sodium phosphate buffer (0.05 M pH 7.0) containing 0.16 M NaCl, 0.015 M sodium azide and

2 3 gelatin at 70° C for 20 min to denature plasma proteins. After the tubes were cooled to room temperature, 0.1 ml antibody (1 :5600 dilution) and radioactive 3H- 49 corticosterone tracer (about 6,000 - 7 ,000 cpm) were added to the tubes. The tubes, with a total volume of 0. 7 ml, were then incubated at 4° C overnight. To separate the bound from the free tracer, 0.2 ml of cold Dextran T 70-charcoal suspension was added to the tubes, mixed and incubated at 4°C for 10 min. The tubes was then centrifuged at 15,000 x g, 4°C for 20 min. The supernatants were decanted into scintillation vials and 5 ml of Ultirna Gold scintillation fluid was added to each vial. The radioactivity of 3H-corticosterone was counted by a Hewlett Packard MINAXY scintillation courter for 2.5 min. The sensitivity of this assay is 0.02 ng/tube, and the intra- and inter-assay variabilities are 4. 5 % and 11. 9 % respectively.

Plasma oxytocin radioimmunoassay

Oxytocin assay was performed by a method modified from Keil ( 1984). Plasma was first extracted before it was used in the radioirnrnunoassay. Briefly, rat plasma (1 ml) was mixed with 2 ml cold acetone (Spectranalyzed) and centrifuged at 2000 rpm,

4°C for 30 min. The supernatant was then added to 5 ml cold petroleum ether and mixed immediately. After centrifugation at 2000 rpm, 4°C for 15 min. the top layer was aspirated and discarded. The remaining solution was dried by blowing air into the tubes in ice. The dried extract was then dissolved in 1 ml assay buffer (0.05 M phosphate buffer pH 7.4, containing 0.125 % bovine serum albumin, 0.01 % sodium azide, 0.001 M EDTA) and was used for the oxytocin radioirnrnunoassay. This assay is a double-antibody assay. The plasma extract (20 or 200 µl) and oxytocin standard

(0-1 ng) were incubated with 0.1 ml rabbit anti-oxytocin serum (1 :50,000 dilution) in a total volume of 0.4 ml assay buffer for 24 hours at 4°C. 1251-oxytocin (3,000 cpm, 50 0.1 ml) was then added to the tubes and incubated for 72 hours at 4°C. To the tubes was then added 0.1 ml goat anti-rabbit )'-globulin (1: 12.5 dilution), followed by 0.1 ml normal rabbit serum (1: 120 dilution). After incubation for 24 hours, the tubes were centrifuged at 15,000 x g, at 4°C for 20 min. The supernatant was decanted and the radioactivity of the pellet was counted for 5 min by a Micromedic 4/200 plus 'Y counter and analyzed from the standard curve using the RIA-AID computer program (Robert

Maciel Associates, Arlington, MA). The concentration of plasma oxytocin was calculated with a correction factor based on the recovery of the extraction.

Radioligand binding assay

Homoi:enate radiolii:and bindint: assays for 5-HT1A receptors in hypothalamus, midbrain and frontal cortex

Tissue preparation

Hypothalamic, midbrain and frontal cortical homogenates were prepared according to the protocol of Leonhardt et al. (1992). Briefly, frozen tissues were placed in a minimum of 25 volumes of ice cold 50 mM Tris-HCl (pH 7.7, 25°C)

containing 0.5 mM EDTA and 10 mM MgS04 and homogenized using a Tekmar

Tissumizer (2 x 5 sec). The homogenate was centrifuged at 37 ,000 x g for 15 min at

4°C. The pellet was resuspended in 30 volumes of buffer, homogenized, incubated at

37°C for 15 min and then centrifuged at 37 ,000 x g for 10 min. Tissue was then washed and centrifuged once more. The tissue for 3H-paroxetine binding was washed for 8 times to remove excess fluoxetine. Finally, the pellet was resuspended to a volume of 30 mg wet wt./ml in cold 50 mM Tris-HCl (pH 7.7) containing 0.5 mM 51

EDTA and 10 mM MgS04 . The protein concentration of the homogenate was measured according to Lowry et al. ( 1951).

3H-8-0H-DPAT binding 3 The H-8-0H-DPAT binding assay was used to determine 5-HT1A receptors in the hypothalamus, midbrain and frontal cortex. Briefly, hypothalamic (1.5 mg wet tissue/tube), midbrain (3 mg wet tissue/tube) or frontal cortical (3 mg wet tissue/tube) homogenates were incubated with 1 ml Tris buffer (50 mM, pH 7.7) containing 10 mM

3 MgS04 , 0.5 mM EDTA, 10 µM , 0.02% ascorbate acid and H-8-0H-DPAT

(159.5 Ci/mmol, 1 mCi/ml, New England Nuclear) in a concentration of 0.75-6.0 nM, at room temperature for 1 hr. Non-specific binding was defined in the presence of 1

µM 8-0H-DPAT. The reaction was stopped by immediate filtration over Whatman

GF/C filters and washed three times with 5 ml of a 50 mM Tris buffer (pH 7.7). The radioactivity of the filters was counted in 5 ml scintillation liquid at 56 % efficiency.

Scatchard analysis was applied for calculation of the Kd and Bmax of 3H-8-0H-DPAT

labelled 5-HT1A receptors.

3 H-5-HT binding for 5-HT1A receptors 3 H-5-HT was used to determine 5-HT1A receptors in the frontal cortex. Briefly,

6 mg (wet wt.) of frontal cortex was incubated with 0.75 - 6.0 nM 3H-5-HT in 2 ml

50 mM Tris buffer (pH 7.7, 25°C), containing 10 mM MgS04, 0.5 mM EDTA, 0.02% ascorbate and 10 µM pargyline for 60 min at room temperature. Non-specific binding was defined in the presence of 1µM8-0H-DPAT. The content of the assay tubes was 52 filtered, washed and analyzed as described for the 3H-8-0H-DPAT binding assay.

3H-paroxetine binding for 5-HT uptake sites

A 3H-paroxetine saturation assay (Battaglia et al.1987) was used to examine 5-

HT uptake sites. The tissue homogenate was incubated for 30 minutes at 37°C and further washed a 8 times in 40 volumes of Tris buffer (pH 7.7, 50 mM Tris, 120 mM

NaCl, 5 mM KCl), followed by centrifugation at 37,000 g at 4°C to wash out fluoxetine and desipramine from the tissues. Preliminary data from our laboratory have indicated 3 that inadequate washes of the tissue resulted in an apparent increase in K0 for H­ paroxetine binding, due to fluoxetine remaining bound to the tissue. However, following 8 washes, as described above, no significant differences were observed between vehicle and fluoxetine pretreated rats. After a final wash, the pellet was resuspended to a volume of 30 mg wet wt/ml in 50 mM Tris buffer (pH 7. 7) containing

120 mM NaCl and 5 mM KCI. 3H-paroxetine (20.3 Ci/mmol, 1 mCi/ml, Du Pont

NEN, Boston, MA) in concentrations of 0.01-0.4 nM was incubated at room temperature with midbrain homogenates (1.5 mg tissue/tube) in 5 ml of 50 mM Tris buffer (pH 7.7), containing 120 mM NaCl and 5 mM KCl, at room temperature for 2 hrs. Non-specific binding was defined in the presence of 1 µM citalopram. The reaction was stopped by immediate filtration over Whatman GF/C filters and the filters were washed three times with 5 ml of a 50 mM Tris buffer (pH 7. 7). The radioactivity of the filters was counted in 5 ml scintillation liquid at 56 % efficiency. The Bmax and

Kd of the 5-HT uptake sites were calculated using Scatchard analysis. 53

Autoradiographic analysis of 3H-8-0H-DPAT binding

Procedure

The from the rats that received saline-challenge were cut into 15 µm coronal sections using a cryostat at -21°C. The sections were thaw-mounted on chromalum/gelatin coated slides and stored at -20°C. Sections were collected from the following levels: frontal cortex (Bregma +3.70 mm), medial hypothalamus (Bregma -

1.80 mm), caudal hypothalamus (Bregma -3.14 mm) and midbrain (Bregma -7.8 mm) according to the atlas of Paxinos & Watson (1986). Sections from these levels were used for autoradiographic analysis.

3 The autoradiographic assay for H-8-0H-DPAT- labeled 5-HT1A receptors was performed as described by Pazos and Palacios (1985). Briefly, after a preincubation in the assay buffer (containing 0.17 M Tris, 4 mM CaCl, 10 µM pargyline and 0.01 % ascorbic acid, pH 7.6), slide mounted sections were incubated with 3H-8-0H-DPAT (2 nM) at room temperature for 1 hour. This concentration of 3H-8-0H-DPAT is equal to its Kd in hypothalamic homogenates (Li et al.1993b). Non-specific binding was defined in presence of the 10-6M 5-HT. Gpp(NH)p-induced inhibition of 3H-8-0H­

DPAT binding was examined by incubating the slides in the presence of 10-5M

Gpp(NH)p. After washing the slides twice with Tris buffer at 4 °C for 5 min, the slides

were dipped in cold H20 and then blow-dried immediately. They then were exposed to tritium-sensitive Hyperfilm-3H for either 2 months or 2 weeks (for sections containing a high density of the binding sites). A set of 3H microscales (Amersham) was exposed to each film together with the slides to calibrate the optical density into 54

fmol/mg tissue equivalent.

The films were developed by a Kodak developing procedure for X-ray films.

Films were placed in a Kodak Developer D-19 for 5 min, followed by 30 sec in Kodak

Indicator Stop Bath solution. Then, the films were transferred into Kodak Fixer for 5

min and subsequently placed in a running water bath for 10 min followed by 5 min in

a water bath containing Kodak Photo-Flo 200. The films were then air-dried.

Data analysis

Autoradiograms were analyzed densitometrically using the NIH image analysis

program for Macintosh computers. The gray scale density readings were calibrated to

fmol/mg tissue equivalent using the [3H]microscales. Brain regions were identified

according to the atlas of Paxinos and Watson (1986), as shown in Fig.10. The layers

of the cortex were identified according to the atlas of Zilles ( 1985). The density of 3H-

8-0H-DPAT binding in each brain region was measured and expressed as fmol/mg tissue equivalent. An area outside of the section was measured as the background of the film, which was subtracted from each measurement in the section. Specific 3H-8-

OH-DPAT binding sites in each brain region were determined by subtracting the non-

specific binding sites from the total binding sites in each region. The percent of

Gpp(NH)p-induced inhibition was calculated using the equation below:

fmol/mg tissue equivalentsp - fmol/mg tissue equivalentopp(NH)p 3 of inhibition - x 100 % fmol/mg tissue equivalentsp

In this equation, fmol/mg tissue equivalentsp represents the specific 3H-8-0H-DPAT 55 binding in the absence of Gpp(NH)p; fmol/mg tissue equivalentapp(NH)p represents the specific binding in the presence of Gpp(NH)p.

The data for a brain region of each rat represents the mean of four adjacent brain sections.

Immunoblot analysis of G proteins

Membrane preparation

The membranes of the hypothalami, midbrains and frontal cortices were prepared as described by Sternweis ( 1984) and Okuhara ( 1996). Briefly, the tissues were homogenized in 1ml50 mM Tris buffer, pH 7.4 containing 150 mM NaCl, 10% sucrose and 0.5 mM phenylmethanesulfonyl fluoride (PMSF; added immediately before homogenization), using a Tekmar tissumizer at a speed of 13,500 rpm for about 10 seconds. After centrifugation at 20,000 x g, 4°C for 60 minutes, pellets were re­ suspended in 50 µl (for the hypothalamus), or 200 µl (for the midbrain and frontal cortex) of 20 mM Tris buffer, pH 8 containing 1 mM ethylenediamine tetraacetic acid

(EDTA), 100 mM NaCl, 1 % sodium cholate and 1 mM dithiothreitol to solubilize the membrane proteins. The re-suspended homogenates were incubated and shaken at 4°C for 1 hour, followed by centrifugation at 100,000 x g, 4°C for 60 minutes. The supernatants were used for G protein measurements. Protein concentrations in the supernatant were measured according to Lowry (1951) using bovine serum albumin as a standard. The protein concentrations in the supernatant were: 1.7-2.2 µglµl for the hypothalamus; 2.0-3.5 µglµl for the midbrain; and 1.6-2.3 µglµl for the frontal cortex. 56 Quantification of G proteins

The solubilized proteins (10-35 µg protein) were resolved by SDS­

polyacrylamide gel electrophoresis, using gels containing 0.1 3 sodium dodecyl sulfate

(SDS), 12% acrylamide/bisacrylamide (30:0.2), 4 M urea and 375 mM Tris, pH 8.4

(Mullaney and Miligan, 1990). Two samples from each treatment group (i.e. control,

fluoxetine 3 days, fluoxetine 7 days, etc.) were loaded on each gel. Each sample was

repeatedly measured on three independent gels. The proteins were then electrophoretically transferred to nitrocellulose membranes. The membranes were

incubated in a solution containing 5 3 non-fat dry milk, 0.05 3 NP40, 50 mM Tris and

150 mM NaCl, pH7.4, and were then washed. The membranes were incubated with polyclonal antisera for Gi1 12 (AS/7, 2500 dilution), Gi3 (Anti-GiaJ• 1 :2000 dilution) and

G0 (GC/2, 1 :2000 dilution) at 4°C overnight. The membranes were then incubated with a secondary antibody (goat anti-rabbit serum 1: 10,000) for 60 minutes. Following 4 washes with 0.05% NP40 in 50 mM Tris and 150 mM NaCl, the membranes were incubated with rabbit peroxidase-antiperoxidase (1: 10,000) for 1 hour. After several washes, the membranes were incubated with the chemiluminescence substrate solution

(LumiGlo) for 1 minute and then exposed to Kodak x-ray film for 10-40 seconds.

Data analysis

Films were analyzed densitometrically using the NIH image analysis program for Macintosh computers. The gray scale density readings were calibrated using transmission step wedge standard (Stouffer Graphic Arts Equipment Co., South Baend,

IN). The integrated optical density (IOD) of each band was calculated as the sum of 57 optical densities of all the pixels within the area of the band outlined. An area adjacent to the G protein bands was used to calculate the background optical density of the film.

The IOD for the film background was subtracted from the IOD for each band. The resulting IOD for each G protein band was then divided by the amount of protein loaded on the corresponding lane. The IOD/ µg protein values obtained from treated rats were divided by the mean IOD/ µg protein values obtained from control rats in same gel to determine the relative amounts of the G proteins. The data for each rat were the means obtained from the three gels. Samples from 6-8 rats were measured for each treatment group.

Characterization of antisera

Recombinant mouse Gil• Gi2 , Gi3 and G0 standards (obtained from CalBiochem) were used to identify the G protein bands and characterize the specificity of antisera.

As Fig.1 shows, Gil protein can be separated from Gi2 protein under our electrophoretic conditions. Therefore, we were able to use an antiserum which recognizes both Gi1 and

Gi2 proteins (AS/7), to measure these Gi proteins independently. Gi3 and G0 proteins were not detected with the AS/7 antiserum. Anti Gi3 serum recognizes at least three bands between 39-45 kd. The Gi3 standard co-migrated with the highest molecular weight band. Therefore, we measured this band as the Gi3 protein. This Gi3 antibody

, did not detect Gi 1 Gi2 or G0 proteins. GC/2, an anti G0 serum, was found to be

selective for G0 protein and did not detect Gil, Gi2 and Gi3 proteins (Fig. 1). 58

AS/7 ~ 45 kDa

--- ~ 32 .6 kDa Gi1 Gi1 Gi2 + Hyp Go Gi3 Gi2

Anti Gi3 ~ 4 5 kDa

~ 32 .6 kDa G;3 + Hyp Go G;, G,2 Gi3 Hyp

Fi2ure 1. Characterization of antibodies against Gi., Gi2, Gu and G0 proteins used in immunoblots. The top panel demonstrates the specificity of AS/7 antibody for the Gi 1 and Gil proteins: The lanes from left to right contain: recombinant Gi1 protein, recombinant Gi2 protein, recombinant Gi 1 plus Gi2 proteins, hypothalamic extract, recombinant G0 protein, and recombinant Gi3 protein. The bottom panel demonstrates the specificity of the UBI Gi3 antibody. The lanes from left to right contain: recombinant Gi3 protein, hypothalamic extract plus recombinant Gi3 protein, hypothalamic extract, recombinant G0 protein, recombinant Gil, and recombinant Gi2 protein. 59

Statistics

The data from the hormone analyses were extrapolated from standard curves using the RIA-AID computer program (Robert Maciel Associates, Arlington, MA).

The data are presented as group means and the standard errors of the means (S.E.M.).

The data from the hormone assays were analyzed by a two-way analysis of variance

(ANOVA) and the data obtained from radioligand binding for the density and affinity

of 5-HT1A receptors and 5-HT uptake sites or from immunoblots for G proteins were analyzed by one-way ANOVA. Group means were compared by Newman-Keuls' or

Duncan's multiple range test as indicated in the text (Steel and Torrie, 1960). A computer program (NWA STATPAK, Portland,OR) was used for all the statistical analyses. CHAPTER IV

LONG-TERM FLUOXETINE, BUT NOT DESIPRAMINE, PRODUCES A

DESENSITIZATION OF HYPOTHALAMIC 5-HTIA RECEPTORS

Summary

The present studies determined whether the hypothalamic 5-HT1A receptor systems are modified by chronic exposure to antidepressants. Hormone responses to

the 5-HT1A agonists 8-0H-DPAT and ipsapirone were used as marker to evaluated the function of 5-HT1A receptors after long-term exposure to two antidepressants, the 5-HT uptake inhibitor fluoxetine and the norepinephrine uptake inhibitor desipramine. In addition, the effects of long-term exposure to fluoxetine and desipramine on the density

and affinity of 5-HT1A receptors in the hypothalamus, midbrain and cerebral cortex were examined. Male rats received fluoxetine (10 mg/kg ip), desipramine (5 mg/kg ip) or saline injections once daily for 21 days. 8-0H-DPAT (0-500 µg/kg sc), or ipsapirone (0-5 mg/kg, ip) were administered 18 hours after the final antidepressant injection and 15 minutes before sacrifice. 8-0H-DPAT and ipsapirone significantly increased plasma ACTH, corticosterone and oxytocin concentration in a dose-dependent manner. Chronic injections of fluoxetine inhibited the ACTH, corticosterone and

oxytocin responses to 8-0H-DPAT or ipsapirone, suggesting reduced 5-HT1A receptor function. In contrast, chronic injections of desipramine did not alter the hormone

60 61 responses to either 8-0H-DPAT or ipsapirone. The density and affinity of 5-HT1A receptors in the frontal cortex or hypothalamus were not altered by either fluoxetine or

desipramine. However, a decrease in the Bmax of 5-HT1A receptors in the midbrain was observed after chronic fluoxetine, but not desipramine. No change in the 5-HT uptake sites was observed after either antidepressant. To verify that the observed effects require prolonged exposure to fluoxetine, rats received a single injection of fluoxetine (10 mg/kg, ip), 3 hours before the injection of 8-0H-DPAT (0-500 µglkg sc). Acute fluoxetine did not reduce any of the hormone responses to 8-0H-DPAT.

In conclusion, the results suggest that chronic, but not acute exposure to fluoxetine

produces a desensitization of hypothalamic 5-HT1A receptors. This effect is not seen in rats chronically exposed to desipramine. The mechanism of the effects of chronic

fluoxetine on 5-HT1A receptor mediated hormone responses is not likely due to reduced

3 density of 5-HT1A receptors, because no changes were observed in H-8-0H-DPAT binding in hypothalamus and cortex. It could be due to changes in signal transduction

system of 5-HT1A receptors. Furthermore, it can not be ruled out that fluoxetine

injections alter the density of 5-HT1A receptors in specific nuclei of the hypothalamus.

Introduction

The present studies examined hormone responses to two 5-HT1A agonists, 8-0H­

DPAT and ipsapirone, to assess the effect of two antidepressants, fluoxetine and

desipramine on 5-HT1A receptor function. Several antidepressants are 5-HT uptake inhibitors with lower affinity for norepinephrine or dopamine uptake sites (e. g fluoxetine, sertraline, paroxetine) (Beasley et al.1992; Levy and Van de Kar, 1992; 62

Perry and Fuller, 1992; Wong et al .1990; Bolden-Watson and Richelson, 1993; Fuller et al.1991). Other antidepressants, such as desipramine, are norepinephrine uptake inhibitors with low affinity for serotonin uptake sites (Richelson, 1991). Regardless of the acute pharmacologic profiles of these antidepressants, the mechanisms of their antidepressant effects remain unclear, particularly because they have to be administered for at least 2-3 weeks before clinical improvement can be observed (Richelson, 1991).

Alterations in 5-HT IA receptors have been suggested to contribute to the clinical improvement observed after long-term exposure to antidepressants (Lesch et al.1991c;

Meltzer, 1990; Richelson, 1991; Newman et al.1992; Stockmeier et al.1992). To

determine whether changes in 5-HT1A receptor systems occur commonly after chronic exposure to two classes of antidepressants, in the present study we compared the effect

of chronic fluoxetine and desipramine on the function of 5-HT1A receptors in the hypothalamus.

Hormone responses to challenges with selective 5-HT agonists can be used as indices of the function of serotonin receptors, both in humans and experimental animals

(Van de Kar, 1989). Activation of 5-HT1A receptors increases the secretion of ACTH, corticosterone and oxytocin (Kelder and Ross, 1992; Van de Kar, 1991; Jorgensen et al.1992; Van de Kar and Brownfield, 1993; Brownfield et al.1992). 8-0H-DPAT is

a selective 5-HT1A agonist (Van Wijngaarden et al.1990; Hoyer and Schoeffter, 1991) that has been shown to increase the secretion of oxytocin, ACTH and corticosterone by

activating 5-HT1A receptors (O'Donnell and Grealy, 1992; Calogero et al.1990; Fuller and Snoddy, 1990; Bagdy et al. 1989; Calogero et al.1989; Koenig et al.1987; Gilbert 63 et al.1988a; Vicentic et al.1996). Ipsapirone is a partial 5-HT IA agonist. Like 8-0H-

DPAT, ipsapirone increases ACTH, corticosterone and oxytocin secretion by activating hypothalamic 5-HT,A receptors (Lesch et al.1991c; Cowen et al.1990; Gilbert et al.1988a; Bagdy, 1995; Bagdy, 1994; Bagdy and Makara, 1994). However, the two

5-HT,A agonists have different chemical structures and thus, they may have different side effects. Since ipsapirone and 8-0H-DPAT have a common pharmacological effect

on 5-HT1A receptors, the common neuroendocrine profiles obtained with these two 5-

HT,A agonists are most likely mediated by activation of 5-HT1A receptors.

In the present studies, we examined the neuroendocrine responses to the 5-HT,A agonists, 8-0H-DPAT and ipsapirone, after chronic exposure to fluoxetine or desipramine. To examine the mechanism of the changes in the 5-HT,A receptor systems induced by the antidepressants, we also measured the affinity and density of

5-HT1A receptors in the hypothalamus, midbrain and cerebral cortex as well as the affinity and density of 5-HT uptake sites. Furthermore, we compared the effects of chronic fluoxetine on the hormone response to 8-0H-DPAT with that of acute treatment with fluoxetine, to determine whether the effects of fluoxetine on the function of 5-HT,A receptors require long-term treatment.

Experimental Protocol

The rats for the chronic experiment were pretreated with either fluoxetine ( 10 mg/kg ip), desipramine (5 mg/kg ip) or saline once daily for 21 days. These doses were observed by de Montigny and Aghajanian (1978) to alter the response of rat forebrain neurons to 5-HT. Furthermore, treatment with these doses has resulted in up- 64

regulation of 5-HT2 receptors in the hypothalamus (Li et al.1993a). 8-0H-DPAT (50,

200, 500 µglkg sc), or ipsapirone ( 1, 2, 5 mg/kg ip) or saline were administered 18 hours after the last injection of the antidepressants. The rats were sacrificed by

decapitation 15 minutes following injection of the 5-HT1A agonists or saline. Trunk blood was collected in centrifuge tubes containing 0.5 ml of a 0.3 M EDTA (pH 7.4) solution. The frontal cortex, hypothalamus and midbrain were dissected immediately after decapitation and frozen in liquid nitrogen. Tissues were stored at -70°C until the

measurement of 5-HT1A receptors and 5-HT uptake sites. In the acute experiment, the rats received a single dose of saline or fluoxetine (10 mg/kg ip) 3 hours before injection of 8-0H-DPAT. The doses of 8-0H-DPAT in the acute experiment were the same as those in the chronic experiment.

Plasma was stored at -70°C until assayed for plasma hormone levels. Plasma

ACTH, corticosterone and oxytocin concentrations were measured by

radioimmunoassay (see Chapter III). The affinity and density of 5-HT1A receptors in the hypothalamus, midbrain and frontal cortex were examined by 3H-8-0H-DPAT binding or 3H-5-HT binding assays. In addition, affinity and density of 5-HT uptake sites in the midbrain were determined by 3H-paroxetine binding (see Chapter Ill for details).

Results

Both 8-0H-DPAT and ipsapirone increased plasma ACTH, corticosterone and oxytocin concentrations in a dose-dependent manner (Fig. 2, 3, 5). Repeated injections of fluoxetine, but not desipramine, significantly inhibited the 8-0H-DPAT-induced 65 increase of plasma ACTH levels (Fig. 2). This inhibition was observed at the 50 µglkg dose of 8-0H-DPAT in fluoxetine pretreated rats (Newman Keuls' test P < 0.01).

Consistent with the ACTH response to 8-0H-DPAT, chronic fluoxetine, but not desipramine, inhibited the ipsapirone-induced increase of plasma ACTH concentration

(Fig. 2). Chronic fluoxetine significantly reduced the ACTH response to the 5 mg/kg dose of ipsapirone (P < 0.01). A single injection of fluoxetine did not significantly alter the ACTH response to 8-0H-DPAT (Fig. 4).

Similar to the ACTH response, chronic fluoxetine, but not desipramine, shifted the corticosterone dose response curve of 8-0H-DPAT to the right, but did not change the maximal response. A significant inhibition of the corticosterone response by chronic fluoxetine was observed at the lowest dose (50 µg/kg) of 8-0H-DPAT (P<

0.05, Newman Keuls' test, Fig. 3). Two-way ANOVA revealed no significant effect of the antidepressants on the ipsapirone-induced increase of plasma corticosterone concentration. However, a significant inhibition by fluoxetine was observed at the 2 mg/kg dose of ipsapirone (Duncan's multiple range test, P < 0.01). In contrast, a single injection of fluoxetine potentiated the corticosterone response to the lowest dose

(50 µg/kg sc) of 8-0H-DPAT (Fig. 4). No significant difference was observed in the corticosterone responses to any dose of ipsapirone after long-term treatment with desipramine (Fig. 3).

Chronic fluoxetine, but not desipramine, inhibited the effect of 8-0H-DPAT on oxytocin secretion as seen by a parallel right-shift in the dose response curves (Fig. 5).

The lower doses of 8-0H-DPAT (50 and 200 µglkg sc) produced significantly lower 66 effects on plasma oxytocin in fluoxetine treated rats (Newman Keuls' test, P < 0.05).

Chronic fluoxetine, but not desipramine, also reduced the oxytocin responses to ipsapirone. Two-way ANOV A showed a significant interaction between the effect of the antidepressants and ipsapirone on plasma oxytocin concentration (F<6.79i=3.062, P

< 0.01). Furthermore, Duncan's multiple range test indicated that chronic fluoxetine significantly inhibited the effect of ipsapirone (5 mg/kg) on plasma oxytocin (P <

0.01). Chronic desipramine did not affect the ipsapirone-induced increase in plasma oxytocin concentration. As can be seen in Figures 5, acute injection of fluoxetine did not inhibit the oxytocin responses to the same doses of 8-0H-DPAT.

As can be seen in Table II, repeated injections of fluoxetine and desipramine did

not significantly alter the density of 5-HT1A receptors in the hypothalamus and frontal cortex. However, chronic fluoxetine, but not desipramine, decreased the density of 3H-

8-0H-DPAT-labeled 5-HT1A receptors in the midbrain. Neither fluoxetine nor desipramine altered the affinity of 3H-8-0H-DPAT binding in any brain region. Also, long-term injections of the antidepressants did not change the density and affinity of 3H­ paroxetine-labeled 5-HT uptake sites in the midbrain. 67

Saline •• Fluoxetine ,--.., Desipramine E 500 • "-... * cr> * ...... _,,a.. 400 ,,•/!~* I 300 1 ' I- / / 1 / * / / .* / u 200 / / // * <( / / / / 0 100 , t E Ul z----c.--r""; •. . I I I I I I 0 / -0 Q_ 0 50 200 500 Dose of 8-0H-DPAT sc) ,...... _ (µg/kg' E 600 ""'cr> 500 •* ...... _,,a.. 400 ///' * I I- 300 u <( ,/l 200 , ,,,. ,,,. t 0 ,, ! ,,.. ,,,. ~ ~ 100 E --=-=-=-=-=-==-=-=-=-=-" - ~ . Q1 0 -0 0... 0 2 5 . . Dose of 1psap1rone (mg/kg, ip)

Figure 2 Effect of chronic exposure to fluoxetine or desipramine on ACTH responses to 8-0H-DPAT (top) or ipsapirone (bottom). The data represent mean± S.E.M of eight to nine rats per group. Two way ANOVA: Main effect of antidepressants: F<2•80>=3.058, P<0.053(top); F<2•82>= 3.8664, P<0.05 (bottom); Main effect of 8-0H-DPAT: F<3•80>=27.56 P<0.01 (top); Main effect of ipsapirone: F<3.82>=29.33 P<0.01 (bottom); Interaction between antidepressants and 5-HT agonists: F<6•80>=2.597 P<0.05 (top); F<6.82>=2.6918 P<0.05 (bottom). *Significant difference from the saline challenged group, P < 0. 01. + significant difference from the saline pretreatment group (injected with the same dose of 5-HT1A agonists), P < 0.05 (Duncan's multiple range test). 68

Saline • Fluoxetine Q) • c Desipramine 0 1 2 * L. • Q) -+-' ,~ (() * / ' ----·--- * ,,. ,,. / _, 9 0 ,,,.-..... ,,.. / (.) "Cl -+-' ,,.. ,/ / / * * L. ""-.,,,, ,I ,I Ol 6 ,I 0 ,I (.) ...._,:::t _... ,,,.,,,. •l. 0 3 E - -- t (() - -- -a 0 0.... 0 50 200 500 Dose of 8-0H-DPAT (µg/kg, sc)

OJ c 15 0 !.... * ClJ 1 2 * _,,• -+-' (/) ,,,.-..... 0 9 '~- )''-- u "O ..... ""-.,,,, !.... 0\ 0 ::1. 6 ~ ,, /// * u .._ ------t.. • 0 3 ~- ____ / t E (fl -0 0 a.. 0 2 5 Dose of ipsapirone (mg/kg, ip)

Figure 3. Effect of chronic exposure to fluoxetine or desipramine on the corticosterone response to 8-0H-DPAT (top) or ipsapirone (bottom). The data represent mean ± S.E.M of eight to nine rats per group. Two way ANOVA: Main effect of antidepressants: F<2.83)=0.912, NS (top); F<2•82)=1.8731, NS (bottom); Main effect of 8-0H-DPAT: F<3•83)=29.40 P<0.01 (top); Main effect of ipsapirone: F<3•82)=18.0487 P<0.01 (bottom); Interaction between antidepressants and 5-HT agonists: F<6•83)=2.262 P<0.0452 (top); F<3•82)=1.1089 NS (bottom). * Significant difference from the saline challenged group P < 0. 05; + significant difference from the saline pretreatment group (injected with the same dose of 5-HT1A agonists), P < 0.05 (Duncan's multiple range test). 69

e SALINE • FLUOXETINE ,,;""., 1000 E '-... 0\ 800 a. '--"' :c 600 I-u <( 400 a t: 200 l"/l a - 0 0... 0 50 200 500 Dose of 8-0H-DPAT (µ,g/kg, sc)

Q) c 20 * 0 t ... ------•1 ---- * L / Q) 1 6 /i* ____ , ->-J -· en ,...... , 0 - 1 2 (.) "C // /* ...... ->-J L C1' 0 ..._,:::l 8 () 0 4 /? E (/) 0 -0 0 50 200 500 0.... Dose of 8-0H-DPAT (µ,g/kg, sc)

Fia=ure 4. Effect of a single injection of fluoxetine on ACTH (top) and corticosterone (bottom) responses to 8-0H-DPAT. The data represent mean ± S.E.M of eight to nine rats per group. Two way ANOV A: Main effect of antidepressants: Fcl,58)=0.267, NS (top); Fcl,58)=4.940, P<0.03 (bottom); Main effect of 8-0H-DPAT: Fc3,58l=52.49 P<0.01 (top); Fc3,58l=27.363 P<0.01 (bottom); Interaction between antidepressants and 8-0H-DPAT: F(3,s8)= 1.119 NS (top); Fc3,58)=2.0225 NS (bottom). * Significant difference from the saline (0 dose of 8-0H­ DPAT) group, P<0.05; +significant difference from the saline pretreatment group (injected with the same dose of 8-0H-DPAT), P<0.05 (Newman Keuls' test). 70

SALi NE ~ E • FLUOXETINE "'-., • Ol 100 OMI * ~~-- * 0...... _,,, 80 • ,t~.* c / .,,.- ',,..... l ·-(J . 60 0 -1-J • * >. 40 * " x / //*t 0 20 ~ ~ ,a / / a ~ .,,, /

E 0 I I I en / .z::.--c--:z::-~1 't1 1 1 a 0 50 200 500 Q_ Dose of 8-0H-DPAT (µ,g/kg I sc) ...... -._ E "'-., 40 cri * ...._,,a.. 30 c • ·-(.) 0 ...... 20 /!* >. x 0 1 0 ·---·-----. 0 - t E (/) 0 -0 0 2 5 Q_ . Dose of 1psap1rone (mg.kg, ip)

Figure 5. Effect of chronic exposure to fluoxetine or desipramine on the oxytocin response to 8-0H-DPAT (top) or ipsapirone (bottom). The data represent mean ± S.E.M of eight to nine rats per group. Two way ANOVA: Main effect of antidepressants: F(2.s4>= 14.429, P < 0.001 (top); F<2•79>=2.618 NS (bottom); Main effect of 8-0H-DPAT: F<3•84>=120.73 P<0.001 (top); Main effect of ipsapirone: F<3.79>=17.7837 P<0.001 (bottom); Interaction between antidepressants and 5-HT agonists: F<6•84>=1.998 P<0.0749 (top); F<6•79>=3.0617 P<0.01 (bottom).* Significant difference from the saline challenge group P < 0. 05; + significant difference from the saline pretreatment group (injected with the same dose of 5-HT1A agonists), P < 0.05 (Duncan's multiple range test). 71

• SALINE ,,...... ,,,. ~-- ...... * • FLUOXETINE * E 80 • • ...... _ //.,~ l. CJl a.. // 1 • ...._,, 60 / * l. c // * / ·-() / 0 / ~ 40 / ,..... /, x /, 0 * 0 20 E • en -0 I ~ 0... 0

0 50 200 500

Dose of 8-0H-DPAT (µg/kg I sc)

Figure 6. Effect of a single injection of fluoxetine on the oxytocin response to 8-0H-DPAT. The data represent mean ± S.E.M of eight to nine rats per group. Two way ANOVA: Main effect of antidepressant: F(1,5s)=5.2487 P<0.026; Main effect of 8-0H-DPAT: F<3•58l=96.959 P<0.001; Interaction between antidepressants and 8-0H-DPAT: F<3•58l= 1.214 NS. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, (Newman Keuls' test). 72

TABLE II

CHANGES IN BMAX OF 5-HT1A RECEPTORS INDUCED BY CHRONIC EXPOSURE TO FLUOXETINE AND DESIPRAMINE

Bmax (finol/Ing protein) Saline Fluoxetine Desipramine 3ff-8-0H-DPAT binding Hypothalainus 196.8 ± 21.89 212.3 ± 29.6 211.1 ± 21.9 (100%) (107.9%) (107.2%) Mid brain 82.19 ± 3.95 64.98 ± 2.71 * 77.7 ± 3.23 (100%) (79.0%) (94.53) Frontal cortex 160.3 ± 8.5 150.7 ± 9.3 155.1 ± 11.5 (100%) (93.7%) (96.7%) 3H-5-HT binding Frontal cortex 158.0 ± 9.0 134.8 ± 11.9 149.0 ± 16.8 (100%) (85.3%) (94.3%) 3H-naroxetine binding Mid brain 650.3 ± 26.7 640.2 ± 30.3 615.2 ± 37.1 (100%) (98.4%) (94.63)

The data represent Inean ± S.E.M of eight rats per group. The nuinbers in parentheses indicate percent of the control group. 73

TABLE III

CHRONIC FLUOXETINE OR DESIPRAMINE DOES NOT CHANGE THE

AFFINITY OF 5-HT1A RECEPTORS AND 5-HT UPTAKE SITES

Kd Saline Fluoxetine Desipramine

3H-8-0H-DPAT bindin2 (nM} Hypothalamus 1.88 ± 0.2 2.39 ± 0.3 2.03 ± 0.3 Midbrain 1.32 ± 0.06 1.39 ± 0.12 1.56 ± 0.13 Frontal cortex 0.94 ± 0.1 0.87 ± 0.05 0.91 ± 0.04 3H-5-HT bindin2 (nM} Frontal cortex 2.0 ± 0.2 1.83 ± 0.12 1.73 ± 0.27 3H-naroxetine bindin2 (,UM} Midbrain 60.1± 2.5 72.0 ± 4.8 58.7 ± 4.0

The data represent mean ± S.E.M of eight rats per group. 74 Discussion

The present results suggest that chronic exposure to fluoxetine produces a desensitization of hypothalamic 5-HT1A receptors. In contrast, chronic injections of desipramine, or an acute injection of fluoxetine do not produce desensitization of the

5-HT1A receptors. These data suggest that the effects of chronic fluoxetine on the 5-

HT1A receptor systems are adaptive changes induced by chronic inhibition of 5-HT uptake. Such reduction in the function of 5-HT1A receptors could be due to down­ regulation of 5-HT1A receptors. However, the inhibitory effects of chronic fluoxetine on hormone responses to 5-HT iA agonists were not accompanied by a significant reduction in the affinity or density of 5-HT1A receptors in the hypothalamus or frontal cortex. These reduced hormone responses are due to unknown mechanisms, possibly alterations in the signal transduction mechanisms, or in the second messenger systems

of 5-HT1A receptors. On the other hand, it cannot be ruled out that the desensitization of 5-HT1A receptors induced by chronic fluoxetine is due to changes in the density of

5-HT1A receptors in specific hypothalamic nuclei, which cannot be detected by the radioligand binding assay in homogenates.

8-0H-DPAT is a selective 5-HT1A agonist with a high affinity for 5-HT1A receptors (pKi = 8.7) (Hoyer and Schoeffter, 1991) and low affinity for other 5-HT receptor subtypes (5-HTrn pKi = 5.75; 5-HT1c pKi = 5.1; 5-HT2 pKi < 5; 5-HT3 pKi

< 5; 5-HT uptake pKi = 6.25) (Van Wijngaarden et al.1990). Several studies have demonstrated that 8-0H-DPAT increases the secretion of ACTH, corticosterone and oxytocin by activating 5-HT1Areceptors (Cowen et al.1990; Gilbert et al.1988a; Koenig 75 et al.1987; Bagdy and Kalogeras, 1993; Li et al.1993 b; Vicentic et al.1996).

Ipsapirone is a partial 5-HT1Aagonist. Like 8-0H-DPAT, ipsapirone increases ACTH, corticosterone and oxytocin secretion by activating hypothalamic 5-HT iA receptors

(Lesch et al.1991c; Cowen et al.1990; Gilbert et al.1988a; Bagdy, 1995; Bagdy, 1994;

Bagdy and Makara, 1994). These two 5-HT1A agonists, ipsapirone and 8-0H-DPAT, have a common pharmacological effect on 5-HT1Areceptors, despite different chemical structures. Therefore, the common neuroendocrine profiles obtained with these two 5-

HT1A agonists are most likely mediated by activation of 5-HT1A receptors. Our observation that chronic exposure to fluoxetine inhibits the ACTH and corticosterone responses to the 5-HT lA agonists is consistent with observations by Lesch et al. (Lesch et al .1991 c). They reported that chronic exposure of humans to fluoxetine inhibited the

ACTH response to ipsapirone (Lesch et al.1991c). These results suggest that chronic exposure to fluoxetine results in desensitization of 5-HT1A receptors both in rats and in humans.

In the present studies, the inhibitory effects of chronic fluoxetine on the oxytocin and ACTH responses to ipsapirone were only observed at a dose of 5 mg/kg and were not as clear as those observed with 8-0H-DPAT. This could be due to the relatively low doses of ipsapirone used in the study. In addition, ipsapirone has a higher efficacy for autoreceptors on the 5-HT neurons in the midbrain than for postsynaptic 5-HT1A receptors in the forebrain (Aghajanian et al.1990; Sprouse and Aghajanian, 1988).

Since the regulation of hormones is mainly mediated by hypothalamic postsynaptic 5-

HT 1A receptors (Gilbert et al.1988b), it is possible that ipsapirone has lower efficacy 76 than 8-0H-DPAT in stimulating hormone release. Bagdy and Kalogeras (1993) observed that plasma oxytocin concentrations were elevated to a higher level by 8-0H­

DPAT (0.2 mg/kg, iv) than by ipsapirone (2 mg/kg, iv). However, ipsapirone can be administered to humans and although is not as useful as 8-0H-DPAT, a challenge with

ipsapirone still has diagnostic value to determine the function of the 5-HT1A receptor system.

The present studies are the first to examine the influence of 5-HT1A receptors on oxytocin secretion. Our results show that 8-0H-DPAT dose-dependently increases plasma oxytocin concentration. Note the similarities in the dose-response curves for plasma ACTH and oxytocin concentrations. Considering the selectivity of 8-0H-DPAT for 5-HT1A receptors (Van Wijngaarden et al.1990), and the dose-dependent effect, the data suggest that the 8-0H-DPAT-induced increase in oxytocin secretion is mediated

by 5-HT1A receptors. Chronic fluoxetine, but not desipramine, produced an inhibition as observed by a parallel shift to the right in the oxytocin dose-response curve. This inhibition of the oxytocin response resembles the inhibition of the ACTH response to

8-0H-DPAT, supporting the hypothesis that alterations in 5-HT1A receptor function might be responsible for this phenomenon.

Since the 5-HT1A agonist-induced hormone responses are directly mediated by activation of cells in the hypothalamus, the lack of change in hypothalamic 5-HT1A receptors after chronic fluoxetine suggests that the decrease of hormone responses to

8-0H-DPAT by chronic fluoxetine is not due to a reduction in the density or affinity

of 5-HT1A receptors. Alternatively, since we have measured receptors in the whole 77 hypothalamus, there could be changes in specific nuclei in the hypothalamus or in other brain regions which could be responsible for the altered hormone responses to 8-0H­

DPAT. Newman et al. (1992) showed that chronic injections of fluoxetine decreased

the function of 5-HT1A receptor systems as measured by the 5-HT-induced inhibition

of forskolin-stimulated adenylyl cyclase activity in the hippocampus. Also, 5-HT1A receptor-mediated hypothermia is decreased after long-term exposure to citalopram and sertraline (Hensler et al.1991) or after electroconvulsive shocks (Stockmeier et al.1992).

These observations suggest that antidepressants produce functional changes in 5-HT1A receptors which are not due to alteration in ligand recognition sites. Thus, it is possible

that the desensitization of 5-HT1A receptors induced by chronic fluoxetine is due to a

dysfunction of the signal transduction system linked to 5-HT1A receptors in the hypothalamus.

An alternative explanation would be that chronic fluoxetine reduces the content of corticotropin releasing factor (CRF), ACTH or oxytocin. This is not likely because chronic exposure to the same doses of fluoxetine and desipramine potentiated the ACTH and oxytocin responses to DOI, a 5-HT2Aizc agonist (Li et al.1993a). These data

suggest that the reduced hormone response to the 5-HT1A agonists is due to reduced 5-

HT tA receptor function rather than due to reduced hypothalamic or pituitary content of releasing factors or hormones.

Although chronic exposure to fluoxetine did not alter the density of 5-HT1A receptors in the hypothalamus and frontal cortex, we observed a reduction of the

density of 5-HT1A receptors in the midbrain after repeated injections of fluoxetine. 78 Since 5-HT neurons are located in the dorsal and median raphe nuclei in the midbrain,

3 the H-8-0H-DPAT-labeledreceptors in the midbrainprobably mainly represent 5-HT1A autoreceptors. Therefore, this result supports the hypothesis that blockade of 5-HT

uptake sites will desensitize 5-HT1A autoreceptors. These data are consistent with the autoradiographic observations by Welner et al. (Welner et al.1989). However, several

other investigators did not observe changes in the 5-HT1A receptors in the raphe nuclei

(Le Poul et al.1995; Hensler et al.1991; Jo las et al.1994).

Most evidence suggests that hormone regulation is mediated by hypothalamic

postsynaptic 5-HT1A receptors (Kelder and Ross, 1992; Gilbert et al.1988b). For example, depletion of 5-HT stores with PCPA does not reduce the ACTH response to

8-0H-DPAT (Gilbert et al.1988b). Therefore, the reduction in the density of 5-HT1A receptors, in the midbrain might not be directly related to the inhibitory effect of

chronic fluoxetine on the hormone responses to 5-HT1A agonists. Neither fluoxetine nor desipramine could alter the density and affinity 5-HT uptake sites in the midbrain, suggesting that the 5-HT nerve terminals in the midbrain are intact. Therefore, the

reduction of 5-HT1A receptors in the midbrain, by fluoxetine, is not likely due to neurotoxic effects of fluoxetine. The lack of alterations in 5-HT uptake sites is consistent with other reports (Cheetham et al.1993; Dewar et al.1993).

In conclusion, chronic exposure to fluoxetine resulted in a desensitization of

hypothalamic 5-HT1A receptors. This effect was not observed in rats chronically injected with desipramine or in rats that received a single injection of fluoxetine. Also,

this effect was opposite from the effect that chronic fluoxetine has on 5-HT2A12c receptor 79 function. The mechanism whereby chronic fluoxetine alters the 5-HT1A receptor- mediated hormone responses is still unclear and it may result from changes in the signal transduction or second messenger systems. CHAPTER V

TIME-COURSE OF FLUOXETINE-INDUCED DESENSITIZATION OF

HYPOTHALAMIC 5-HT1A RECEPTORS

Summary

We examined the time-course of fluoxetine-induced desensitization of 5-HT1A receptors in the hypothalamus. Our previous studies indicated that daily injections of the 5-HT uptake inhibitor and antidepressant drug fluoxetine for 21 days produced

desensitization of hypothalamic 5-HT1A receptors, without altering their density or

affinity. This desensitization was evident from a reduced ability of the 5-HT1A agonist

8-0H-DPAT to elevate plasma levels of ACTH, corticosterone and oxytocin. In the present study, we further studied the time-course of the desensitization of hypothalamic

5-HT1A receptors induced by repeated injections of fluoxetine. To begin to examine the

mechanism of 5-HT1A receptor desensitization, the density of 5-HT1A receptors in specific hypothalamic nuclei was examined using autoradiographic analysis of 3H-8-0H­

DPAT binding. Furthermore, the coupling of 5-HT1A receptors to their G proteins was studied by examining Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding.

Finally, levels of Gi and G0 proteins in the hypothalamus, midbrain and frontal cortex were determined using immunoblots. Daily injections of fluoxetine (10 mg/kg/day) for

3, 7, 14 or 22 days gradually shifted to the right the dose-response curve of the effects

80 81 of 8-0H-DPAT on plasma ACTH, corticosterone and oxytocin. This was particularly evident from the inhibition of the hormone responses to the low (50 µg/kg sc) dose of

8-0H-DPAT. Fluoxetine reduced the maximal elevation in plasma oxytocin but not

ACTH and corticosterone after injection of higher (200 and 500 µg/kg sc) doses of 8-

0H-DPAT. An examination of the time-course revealed a partial reduction of the

ACTH and oxytocin responses to 8-0H-DPAT (50 µg/kg sc) after 3 days and a maximal reduction of all hormone responses after 14 days of fluoxetine injections.

After 14 days of fluoxetine, the ACTH and oxytocin responses to the 50 µg/kg dose of

8-0H-DPAT were virtually blocked. Repeated injections of fluoxetine did not alter the

density of 5-HT1A receptors and did not reduce their coupling to G proteins in any brain

region. This result suggests that the desensitization of 5-HT1A receptors induced by

repeated injections of fluoxetine is not due to changes in the density of 5-HT1A receptors in the hypothalamus. On the other hand, the hypothalamic levels of Gil and Gi3 proteins were significantly reduced after 7 and 14 days of fluoxetine injections. The levels of

G0 and Gi2 proteins in the midbrain were significantly decreased after 3 days and remained reduced for the duration of fluoxetine injections. Fluoxetine did not reduce

the concentrations of Gi or G0 proteins in the frontal cortex at any time. The similarity in time-course between fluoxetine-induced reductions in hormone responses to 8-0H­

DPAT and the reduction in hypothalamic levels of Gi1 and Gi3 proteins suggests that the reduction hypothalamic levels of Gi3 and/or Gi1 proteins plays a role in the gradual

desensitization of 5-HT1A receptors induced by fluoxetine. Furthermore, the earlier

onset of decreased levels of G0 proteins in the midbrain may be involved in the reported 82 desensitization of somatodendritic 5-HT1A autoreceptors in the raphe nuclei, after three daily injections of fluoxetine (Le Poul et al.1995). In conclusion, repeated injections

of fluoxetine produce a delayed and gradual desensitization of hypothalamic 5-HT1A

receptors. The mechanism of desensitization of hypothalamic 5-HT1A receptors may involve decreased levels of Gil and Gi3 proteins, but not a change in the density of 5-

HT IA receptors.

Introduction

Our previous studies demonstrated that daily injections of fluoxetine for three

weeks reduce the ACTH and oxytocin responses to two different 5-HT1A agonists, 8-

0H-DPAT and ipsapirone. A single injection of fluoxetine did not inhibit the hormone responses to 8-0H-DPAT. These results suggest that adaptive mechanisms are

triggered by fluoxetine which lead to the desensitization of hypothalamic 5-HT1A receptors. The present study was designed to determine the time-course of fluoxetine-

induced desensitization of 5-HT1A receptor systems. The desensitization of

hypothalamic 5-HT1A receptors was determined functionally from the reduction in

hormone responses to the 5-HT1A agonist 8-0H-DPAT.

Because no changes were observed in the density and affinity of 3H-8-0H­

DPAT-labelled 5-HT1A receptors in the hypothalamic homogenate, we hypothesized that

the desensitization of hypothalamic 5-HT1A receptors may be due to alterations in their

signal transduction mechanisms, or due to changes in the density of 5-HT1A receptors in specific hypothalamic nuclei. In the present study, we determined the time-course

of effect of fluoxetine on the G proteins that are coupled to 5-HT1A receptors and on 83 the density of 5-HT1A receptors in hypothalamic nuclei and other brain regions. The

signal transduction of 5-HT1A receptors was evaluated by examining the levels of Gi and

G0 proteins which may be coupled to the 5-HT1A receptors. In addition, the degree of

Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding was used to determine the

coupling of 5-HT1A receptors to their G proteins. The density of 5-HT1A receptors in hypothalamic nuclei was examined by autoradiographic analysis of 3H-8-0H-DPAT binding.

5-HT1A receptors are G protein-coupled receptors. According to the model proposed by Lefkowitz et al. (1976), G protein-coupled receptors exist in high

(coupled) and low (uncoupled) affinity states with respect to agonists. In general, the low affinity of agonists for the uncoupled state of receptors precludes labeling of these receptors by radiolabeled agonists. The coupled receptor-G protein configuration only exists while GDP is bound to the a-subunit of the G protein. In the presence of an agonist, GDP will be exchanged with GTP on the a-subunit of G proteins, resulting in dissociation of the G proteins from the receptors and a return of the receptors to the low affinity-state. Gpp(NH)p is a non-hydrolyzable GTP analog, that binds irreversibly

to G proteins and dissociates them from their receptors. In the case of 5-HT1A

receptors, in the presence of Gpp(NH)p, the high affinity-state 5-HT1A receptors will be converted into the low-affinity state, resulting in inhibition of the binding of an agonist, such as 8-0H-DPAT. Therefore, the percent inhibition of 3H-8-0H-DPAT

binding, in the presence of Gpp(NH)p, represents the percent of 5-HT1A receptors that are coupled to their G proteins. In the present studies, we determined the degree of 5- 84 HT IA receptors coupling in nuclei of the hypothalamus and in other brain regions by examining the inhibition of 3H-8-0H-DPAT binding with Gpp(NH)p.

5-HT1A receptors are subclassified into somatodendritic and postsynaptic receptors. Somatodendritic 5-HT1A autoreceptors are located on 5-HT neurons in the raphe nuclei and function as feedback regulators of serotonergic neurotransmission.

Activation of 5-HT1A autoreceptors will inhibit the firing-rate of 5-HT neurons and consequently, decrease 5-HT release from nerve terminals. Postsynaptic 5-HT1A receptors are distributed in most forebrain neurons. The function of postsynaptic 5-

HT1A receptors depends on the function of the target neurons. Activation of 5-HT1A receptors in the hypothalamic paraventricular nucleus increases the secretion of corticotropin releasing hormone (CRH), leading to increased release of ACTH and corticosterone and also increases the secretion of oxytocin (Fletcher et al.1996;

Critchley et al.1994a; Van de Kar and Brownfield, 1993; Gilbert et al.1988a; Bagdy and Makara, 1994; Bagdy and Kalogeras, 1993). Other physiological roles for 5-HT1A receptors in this limbic brain area include food intake, mood changes and sexual behaviors (Uphouse et al.1994b). Therefore, it is important to know the distribution of 5-HT1A receptors in the different regions of the hypothalamus. While autoradiographic information exists on the distribution of 5-HT1A receptors in several brain regions, little has been known regarding the distribution of 5-HT1A receptors in nuclei of the hypothalamus. In the present study, we measured the effect of repeated injections of fluoxetine on the density of 5-HT1A receptors in hypothalamic nuclei and other brain regions using an autoradiographic analysis of 3H-8-0H-DPAT binding. This 85 study not only investigated whether repeated injections of fluoxetine induce a down-

regulation of 5-HT1A receptors in specific hypothalamic nuclei, but also provided a map

of the distribution of 5-HT1A receptors in the hypothalamus and in several other brain regions.

5-HT1A receptors are coupled to G0 and/or Gi proteins (Raymond et al.1993;

Fargin et al.1991; Sprouse and Aghajanian, 1988; Emerit et al.1990). Somatodendritic

5-HT1A autoreceptors in the raphe nuclei are coupled to G0 proteins, and lead to an increase in the opening of K+ channels (Sprouse and Aghajanian, 1988). Activation of

5-HT1A receptors that are coupled to Gi proteins will inhibit the activity of adenylyl cyclase and, consequently, decrease the intracellular concentration of cAMP (Varrault

et al.1994). 5-HT1A receptors have a high affinity for Gi proteins; the rank order of

affinity is Gi3 > Gi1 > Gi2 > G0 proteins (Raymond et al.1993). In the present study,

the effects of repeated injections of fluoxetine on the levels of Gi1, Gi2, Gi3 and G0 proteins in the hypothalamus, midbrain and frontal cortex were examined using immunoblot analysis.

Experimental Protocol

Rats received daily injections of fluoxetine ( 10 mg/kg, ip) for 3, 7, 14 or 22 days. A control group received saline for 22 days. Eighteen hours after the last injection, the rats received a challenge injection of 8-0H-DPAT (0, 50, 200 or 500

µglkg, sc) and were decapitated 15 minutes after the injection of 8-0H-DPAT. Trunk blood was collected in centrifuge tubes containing 0.5 ml of 0.3M ethylenediamine tetraacetic acid (EDTA; pH 7.4). After centrifugation at 2,500 rpm, 4°C for 15 min, 86 the plasma aliquots were stored at -70°C for hormone assays. The brains were collected for autoradiographic analysis of 3H-8-0H-DPAT binding or were dissected and the hypothalami, midbrains and frontal cortices were stored at -7CJ'C for immunoblot analysis of G proteins (see Chapter III for detail).

Plasma ACTH, corticosterone and oxytocin concentrations were measured by radioimmunoassay as detailed in Chapter III.

Results

Hormone responses to 8-0H-DPAT

8-0H-DPAT increased plasma ACTH concentrations in a dose-dependent manner

(Fig. 6). The ACTH response to the 50 µg/kg dose of 8-0H-DPAT was significantly decreased by repeated injections of fluoxetine (Top panel of Fig. 6). This reduction appeared within 3 days (p < 0. 05) and reached a maximal reduction after 14 daily injections of fluoxetine (p < 0. 01, bottom panel of Fig. 6). Also, fluoxetine injections for 22 days significantly reduced the ACTH response to the higher dose (200 µg/kg sc) of 8-0H-DPAT. However, repeated injections of fluoxetine did not alter the maximal

ACTH response to the highest dose of 8-0H-DPAT (500 µg/kg).

Consistent with the ACTH data, 8-0H-DPAT also increased plasma corticosterone levels in a dose dependent manner (Fig. 7). The corticosterone response to the 50 µg/kg dose of 8-0H-DPAT was not significantly reduced after 3 daily injections but started to be significantly reduced after 7 daily injections of fluoxetine

(bottom panel of Fig. 7). The maximal corticosterone response to high doses of 8-0H­

DPAT was not influenced by repeated injections of fluoxetine. 87 8-0H-DPAT significantly increased plasma concentrations of oxytocin in a dose- dependent manner (Fig. 8). The dose-response curve of oxytocin was significantly shifted to the right after 3 days of fluoxetine injections (Fig. 8, top panel). The reduction of the oxytocin response to the 50 µg/kg dose of 8-0H-DPAT was maximal after 14 days of fluoxetine injections (Fig. 8 bottom panel). The oxytocin responses to all three doses of 8-0H-DPAT were decreased by fluoxetine. However, the degree of inhibition by fluoxetine was greater in the rats challenged with the 50 µg/kg dose of

8-0H-DPAT than in the rats that received higher doses (200 or 500 µg/kg) of 8-0H­

DPAT. 88

e SALINE .&. FLUOXETINE 3 DAYS Y FLUOXETINE 7 DAYS + FLUOXETINE 14 DAYS ...... E 1200 • FLUOXETIN E 22 DAYS * ~ 1000 c...... 800 * ~ 600 u <( 400 ' <( ::'2 200 Vl <( _J 0 Q_ 0 50 200 500 Dose of 8-0H-DPAT (µ,g/kg s.c)

C=:J SALi NE E 800 * '-.. ~ 8-0H-DPAT (50 ,ug/kg sc) CJ'I ~ 600 I t; 400 t <(

<( :::?: 200 (/) <( _J 0 a.. VHE 7 14 22 Days of fluoxetine

Fi1mre 7. Daily injections of fluoxetine inhibit the effect of 8-0H-DPAT on plasma ACTH. Top: dose-response curves; Bottom: time-course of the reduction observed with a 8-0H-DPAT dose of 50 µg/kg sc. The data represent mean± S.E.M. of 8-12 rats per group. Two way ANOVA: Main effect of fluoxetine: F(4, 160)=2.69996

P <0.05; Main effect of 8-0H-DPAT: F(3, 160)=95.554 P < 0.001; Interaction between fluoxetine and 8-0H-DPAT: F( 12, 160)=4.836 P<0.001. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, P < 0.05; +Significant difference from the saline (0 dose of fluoxetine) group, P<0.05 (Newman Keuls' multiple range test) 89

e SALINE A FLUOXETINE 3 DAYS 'Y FLUOXETINE 7 DAYS + FLUOXETINE 14 DAYS • FLUOXETINE 22 DAYS c 30 0 I.... 25 Q) * ~--~1* !*~+~ ---;?~·~- * * .J'<" 1 .·.. : 0 / E 5 - ...... ~f.,.. ...,,...... t (/) 0 0 /( e - n e Ir I a... 0 50 200 500 Dose of 8-0H-DPAT (µg/kg s.c)

cu c 25 * D SALINE 0 8-0H-OPAT (50 µg/kg sc} I.... ~ ......

Figure 8. Daily injections of fluoxetine inhibit the effect of 8-0H-DPAT on plasma corticosterone. Top: dose-response curves; Bottom: time-course of the reduction observed with a 8-0H-DPAT dose of 50 µg/kg sc. The data represent mean

± S.E.M. of 8-12 rats per group. Two way ANOVA: Main effect of fluoxetine: F<4•

133)= 1.461 NS; Main effect of 8-0H-DPAT: F<3• 133)=85.245 P <0.001; Interaction between fluoxetine and 8-0H-DPAT: F<12. 133l=2.14 P<0.05. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, P<0.05; +Significant difference from the saline (0 dose of fluoxetine) group, P<0.05 (Newman Keuls' multiple range test) 90

e SALINE ..t.. FLUOXETINE .3 DAYS 'Y FLUOXETINE 7 DAYS + FLUOXETINE 14 DAYS * E 120 • FLUOXETINE 22 DAYS e '-... g: 100 *• ----- L z 80 u :/~~· I­ 60 >­ - /~/·~·*Tt * t x / .. ~ 0 40 // ~·~ t / ,,;' <( ..t..' <'!' 20 ,,,,,,,,,,,..,,,. .(. ~ (/) <( 0 ~· _J a... 0 50 200 500 Dose of 8-0H-DPAT (µg/kg s.c)

E * ~ SALINE 6; 70 ~ 8-0H-DPAT (50 µ.g/kg sc) a.. ..._., 6 0 z 50 g 40 >:: 30 x 0 20 <( 1 0 (/)~ 0 <( _J VHE 7 1 4 22 a.. Days of fluoxetine

Fi1:ure 9. Daily injections of fluoxetine inhibit the effect of 8-0H-DPAT on plasma oxytocin. Top: dose-response curves; Bottom: time-course of the reduction observed with a 8-0H-DPAT dose of 50 µg/kg sc. The data represent mean± S.E.M. of 8-12 rats per group. Two way ANOVA: Main effect of fluoxetine: F<4• 150)=23.5 P<0.001; Main effect of 8-0H-DPAT: F(3, 150)=274.5 P<0.001; Interaction between fluoxetine and 8-0H-DPAT: Fc 12, 150)=3.974 P<0.001. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, P<0.05; +Significant difference from the saline (0 dose of fluoxetine) group, P<0.05 (Newman Keuls' multiple range test) 91

Autoradiographic analysis of 3H-8-0H-DPAT binding

A high density of 5-HT1A receptors was observed in several nuclei of the hypothalamus and amygdala as well as in the hippocampus, dorsal raphe nucleus and in several layers of the frontal cortex (Figures 10 and 11). In the hypothalamus, the

density of 5-HT1A receptors was markedly different among several nuclei, with differences of ten-fold between the highest and lowest densities in the regions measured

(Table IV and Fig. 10). For example, the density of 5-HT1A receptors in the lateral hypothalamic area was 6.8 ± 0.8 (fmol/mg tissue equivalent) while the density in the central sub-division of the ventromedial nucleus was 62.8 ± 4.1 (fmol/mg tissue equivalent; p < 0.01). In the ventromedial nucleus, the central sub-division contained

the highest density of 5-HT1A receptors while the ventrolateral sub-division of the

ventromedial nucleus contained a significantly higher density of 5-HT1A receptors than the dorsomedial subdivision of the ventromedial nucleus (38.0 ± 2.0 vs 29.1 ± 2.7,

fmol/mg tissue equivalent; P<0.05; Table IV). The density of 5-HT1A receptors in the parvocellular sub-division of the paraventricular nucleus (14.0 ± 1.8 fmol/mg tissue equivalent) was not significantly different from that in the magnocellular region (9.1 ±

1.4; fmol/mg tissue equivalent; Table IV) but both were significantly higher than the

lateral hypothalamic area (p < 0.05). The density of 5-HT1A receptors in the dorsomedial hypothalamic nucleus was significantly higher than the density in the two sub-divisions of the paraventricular nucleus. The lateral hypothalamic area contained

the lowest density of 5-HT1A receptors in the hypothalamus.

A very high density of 5-HT1A receptors was found in the hippocampal CAl and 92 dentate gyms (Fig. 11 and Table IV), in the dorsal raphe nucleus, and layers 5 and 6

of the frontal cortex (Fig. 11 and Table IV). Great differences in density of 5-HT1A

receptors were observed in the amygdala. A low density of 5-HT1A receptors was observed in the central amygdaloid nucleus (13.3 ± 2.3 fmol/mg tissue equivalent) and the posterior part of the lateral amygdaloid nucleus (16.9 ± 2.5 fmol/mg tissue equivalent), while a high density was observed in the basomedial amygdaloid nucleus

(65.5 ± 4.1 fmol/mg tissue equivalent). In the cortex, the lowest density of 5-HT1A receptors was observed in layers 1-3 of the parietal cortex, while high densities were observed in layers 5 and 6 of the cingulate and frontal cortices (Table IV).

Table IV shows the effects of repeated injections of fluoxetine on specific 3H-8-

0H-DPAT binding in several brain regions. No change was observed in the density

3 of H-8-0H-DPAT-labelled 5-HT1A receptors binding sites in any brain regions after daily injections of fluoxetine. 93 Medial hypothalamus Caudal hypothalamus

Fi1rnre 10. Autoradiogram of 3H-8-0H-DPAT binding and Gpp(NH)p­ induced inhibition of 3H-8-0H-DPAT binding in the hypothalamus. Medial hypothalamus (left column) and caudal hypothalamus (right column) were examined. The top panel represents the total 3H-8-0H-DPAT binding; the middle panel represents 3H-8-0H-DPAT binding in the presence of Gpp(NH)p (1Q-5M); the bottom panel represents non-specific binding defined by 3H-8-0H-DPAT binding in the presence of 10-6M of 5-HT. Abbreviations: ABL: Basolateral amygdaloid nucleus; ABM: Basomedial amygdaloid nucleus; AC: Central amygdaloid nucleus; AHN: Anterior hypothalamic nucleus; ALP: Lateral amygdaloid nucleus, posterior region; AM: Medial amygdaloid nucleus; CAl-3: Field CAl, CA2 and CA3 of ammon's horn in the 94 hippocampus; DG: Dentate gyrus (hippocampus); DMH: Dorsomedial hypothalamic nucleus; LH: Lateral hypothalamic area; PAR 1: Parietal cortex, area 1; PVNm: Paraventricular hypothalamic nucleus, magnocellular region; PVNp: Paraventricular hypothalamic nucleus, parvocellular region; VMHC: Ventromedial hypothalamic nucleus, central region; VMHDM: Ventromedial hypothalamic nucleus, dorsomedial region; VMHVL: Ventromedial hypothalamic nucleus, ventrolateral region 95 Midbrain Frontal cortex

Fi1:ure 11. Autoradiogram of 3H-8-0H-DPAT binding and Gpp(NH)p­ induced inhibition of 3H-8-0H-DPAT binding in the midbrain (left column) and frontal cortex (right column). The top panel represents the total 3H-8-0H-DPAT binding; the middle panel represents 3H-8-0H-DPAT binding in the presence of Gpp(NH)p (1Q-5M); the bottom panel represents non-specific binding defined by 3H-8- 0H-DPAT binding in the presence of 10-6M of 5-HT. Abbreviations: CG3:Cingulate cortex, area 3; DR:Dorsal raphe; Fr2:Frontal cortex, area 2; MR:Medial raphe 96 Table IV

REPEATED INJECTIONS OF FLUOXETINE DO NOT ALTER THE

DENSITY OF 5-HT1A RECEPTORS

3 Density of H-8-0H-DPAT - labeled 5-HT1A receptors (fmol/mg tissue equivalent)

Brain regions Nuclei Saline 3 days 7 days 14 days 22 days Cortex Fr2 layer 1-3 26.2± 1.3 25.0± 1.5 25.0±2.5 25.2±1.9 26.8±2.5 layer 5 79.3±4.5 78.4±4.7 79.6±4.2 77.9±3.5 84.6±4.0 layer 6 64.1±4.7 61.8±5.4 64.9±4.4 62.3±3.8 68.7±4.4 ------~------CG3 layer 1-3 44.3 ±5.8 41.1 ±5.4 42.4±5.3 42.2±3.4 48.5±2.5 layer 5 65.3±6.7 65.8±8.2 65.9±7.2 65.4±5.1 70.2±3.4 layer 6 57.4±5.1 57.2±7.0 58.1±5.81 57.4±3.4 61.7±3.7 ------~------PARl layer 1-3 13.5±2.0 14.7±1.8 13.6± 1.8 16.2±1.1 15.0± 1.6 layer 4 28.8± 1.3 26.9±2.2 28.1±1.8 29.5±3.0 31.0±2.0 layer 5-6 46.5±2.0 42.4±3.1 42.6±3.3 42.9±5.2 46.5±2.9 Hypothalamus PVNm 9.1±1.4 8.0±1.0 10.4± 1.4 9.6±1.4 10.9±1.3 PVNp 14.0± 1.8 14.2±1.1 16.5±1.6 15.2±1.1 15.8± 1.2 AHN 18.5±2.0 18.6±0.9 20.8±1.9 20.5±1.5 21.3±1.3 LH 6.8±0.8 6.1±0.7 6.5±1.1 6.3±0.4 7.2±0.7 VMHDM 29.1±2.7 24.1±1.1 28.7±1.8 28.2±2.1 27.7±1.3 VMHC 62.8±3.6 54.9±5.3 58.0±3.7 57.3±7.0 54.3±3.6 VMHVL 38.0±2.0 32.3±3.4 33.5±1.7 34.5±3.8 31.1±2.9 DMH 22.4±2.6 19.7±2.1 21.1±1.0 18.4±0.8 18.1±1.1 Amygdala AC 13.3±2.3 13.2±1.9 13.3±2.5 14.7±1.6 14.7±1.9 APL 16.9±2.5 16.8±1.4 16.5±2.3 16.9±1.7 17.8±2.9 ABL 32.2±3.2 28.2± 1.1 31.7±1.4 29.1±2.0 31.1±3.1 ABM 65.5±4.1 57.3±4.5 65.3 ±3.3 61.1±6.5 62.5±4.3 AM 39.4±2.1 36.0±2.2 41.1±1.9 38.0±4.2 38.3±2.4 97

TABLE IV-Continued

Density of 3H-8-0H-DPAT - labeled 5-HTlA receptors (fmol/mg tissue equivalent)

Brain regions Nuclei Saline 3 days 7 days 14 days 22 days

Hippocampus CAl 103.6±7.1104.5±6.4 114.6±8.2 114.9±8.4 117.6±5.8 CA2 19.5±1.3 19.0±1.0 19.9±1.3 22.4±2.3 19.5±1.7 CA3 49.0±3.0 44.9±5.5 51.3±3.7 47.9±6.0 48.2±5.2 DG 126.4±9.7 127.0±6.7 135.1±8.2129.2±7.2133.8±4.5 Mid brain DR 79.2±5.5 59.6±8.3 79.7±4.3 66.0±6.8 80.1±4.9 MR 35.5±3.9 25.9±3.9 35.4±3.4 23.3 ±3.0 24.1±3.8

The data represent mean ± S.E.M. of 5-6 rats per group.

ABL: Basolateral amygdaloid nucleus ABM: Basomedial amygdaloid nucleus AC: Central amygdaloid nucleus AHN: Anterior hypothalamic nucleus ALP: Lateral amygdaloid nucleus, posterior region AM: Medial amygdaloid nucleus CAl-3: Field CAl, CA2 and CA3 of ammon's horn in the hippocampus CG3: Cingulate cortex, area 3. DG: Dentate gyms (hippocampus) DMH: Dorsomedial hypothalamic nucleus DR: Dorsal raphe Fr2: Frontal cortex, area 2 LH: Lateral hypothalamic area MR: Medial raphe PAR 1: Parietal cortex, area 1 PVNm: Paraventricular hypothalamic nucleus, magnocellular region PVNp: Paraventricular hypothalamic nucleus, parvocellular region VMHC: Ventromedial hypothalamic nucleus, central region VMHDM: Ventromedial hypothalamic nucleus, dorsomedial region VMHVL: Ventromedial hypothalamic nucleus, ventrolateral region 98 Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding

Gpp(NH)p inhibited 3H-8-0H-DPAT binding in all brain regions examined

(Figures 10 and 11). However, the degree of inhibition varied from 143 to 853 between several brain regions (Fig. 12). For example, Gpp(NH)p produced 853 inhibition of 3H-8-0H-DPAT binding in the central nucleus of the amygdala, but only

173 inhibition in the dorsal raphe nucleus (Fig 12). In the hypothalamus, the greatest inhibition was observed in the dorsomedial nucleus (61 3), while the lowest degree of inhibition was observed in the central sub-division of the ventromedial nucleus (143; see Fig. 12). In the paraventricular nucleus, a greater inhibition was observed in the parvocellular sub-division (41 %) than in the magnocellular sub-division (20. 7 %)

(Fig.10, 12).

As Table V shows, fluoxetine did not alter the Gpp(NH)p-induced inhibition of

3H-8-0H-DPAT binding in any brain region after the daily injections. 99

100 Hypothalamus

(J"l 80 c 60 "D c 40 _o 20 I- <( 0 o_ E a. z J: :::t u ...J J: z <.) J: ....I Cl J: > ::ii > <( J: ::!!: :c Cl 0 ::!!: a.. ::ii a.. ::!!: > > I 100 > I Other brain regions 0 80 I 60 CX) I 40 I n 20 '+-- 0 0 u a...... I ::ii ::it ..- C'll l"l C!) 0:: 0:: < ...J m CD < < < < a a ::!!: < < < 0 0 <.) c 0 IE ~ I IE ~1 Amygdolo Hippocampus +-' 100 _o 80 ...c Frontal cortex ..c 60

~ 40 20

0 I') lO . .... >. It) >. >. I.. >. I...... 0 0 0 v 0 0 . ...J ...J >. ...J ...J >. ...J >. 0 0 0 0 ...J ...J ...J ...J I EFr 2 I I ECG 3 I I PAR 2 I

Figure 12 (See next page for figure legend) 100 Figure 12. Degree of Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding varies between brain regions. The data are represented as mean ± S.E.M. The abbreviations are the same as listed in Table V. 101 TABLE V

REPEATED INJECTIONS OF FLUOXETINE DO NOT ALTER THE PERCENT

OF G PROTEIN - LINKED 5-HT1A RECEPTORS

Brain regions Nuclei % Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT Saline 3 days 7 days 14 days 22 days Cortex Fr2 layer 1-3 35.8±3.5 33.7±6.7 34.3±5.8 33.4±7.1 31.8±3.3 layer 5 21.2±2.1 20.8±4.5 15.3±3.8 22.1±2.9 25.4±4.7 24.2±2.2 18.6±3.8 17.3±3.6 24.1±2.2 28.2±5.3 ------~'!Y-~E-~- ~------CG3 layer 1-3 31.1 ±2.2 25.3±0.7 18.6±5.8 32.5±7.7 35.2±7.0 layer 5 30.0±2.0 25.6±3.1 21.2±5.0 34.4±3.9 35.7±3.1 21.3±2.6 21.2±3.2 19.4±5.8 27.5±3.0 32.6±3.0 ------~'!Y-~E-~- ~------PAR 1 layer 1-3 42.9±7.6 50.7±6.7 40.2±6.4 49.0±4.3 46.7±4.9 layer 4 34.7±4.8 35.7±2.0 28.4±2.1 33.5±3.3 38.7±3.5 layer 5-6 34.3±4.5 35.8±2.6 27.9±2.8 29.0±4.8 36.8±3.6 Hypothalamus PVNm 41.0±7.7 32.7±8.3 42.7±8.2 28.4±8.1 33.1 ±8.2 PVNp 20.7±7.7 27.3±5.8 31.0±5.7 30.7±6.5 33.5±4.6 AHN 19.1±7.4 30.4±4.7 29.4±4.8 33.6±4.8 32.6±4.2 LH 22.6±5.6 30.7±7.6 31.3±4.7 36.2±7.4 41.4±5.9 VMHDM 25.0±3.9 26.2±4.1 21.1 ±4.5 26.6±4.8 18.3±1.3 VMHC 14.3±5.5 27.7±5.6 19.0±4.7 17.4±7.3 16.3±4.8 VMHVL 35.6±4.8 40.7±5.5 37.8±5.7 36.7 ±5.8 35.6±5.4 DMH 61.4+5.6 61.0+3.0 57.7+5.2 56.2+2.8 52.4±2.3 Amygdala AC 85.5±3.5 85.0±1.3 85.4± 1.9 88.2±0.7 85.0±0.9 ALP 54.1±3.5 56.9±1.9 59.2±3.8 66.0±3.8 54.2±2.7 ABL 44.8±3.6 44.4±5.4 46.0±4.7 42.8±2.5 40.1 ±5.4 ABM 28.4±3.1 31.9±4.3 33.7±2.9 27.6±6.1 32.6±2.8 AM 30.0+5.1 36.2+4.9 35.9+4.9 31.3±6.4 33.5+4.4 102

TABLE V-continued

Brain regions Nuclei % Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT Saline 3 days 7 days 14 days 22 days Hippocampus CAI 20.0±4.0 I2.8±6.0 I7.I ±2.8 13.8±2.2 22.2±3.6 CA2 39.6±3.8 39.I±4.I 38.4±4.2 35.4±6.0 41.0±5.6 CA3 23.6±4.7 28.I ±5.I 25.4±6.8 23. I ±6.2 35.8±5.5 DG 21.0±1.7 20.6±3.6 23.5 ±5.3 20.7±4.5 31.0±3.3 Mi dbrain DR I7.4±4.3 I2.6±7.4 21.3±4.I 26.9±2.4 25.6±3.8 MR 43.7+4.9 47.7+4.5 55.4+3.3 34.2+2.3 38.6+5.5

The data represent mean ± S.E.M. of 5-6 rats per group.

ABL: Basolateral amygdaloid nucleus ABM: Basomedial amygdaloid nucleus AC: Central amygdaloid nucleus AHN: Anterior hypothalamic nucleus ALP: Lateral amygdaloid nucleus, posterior region AM: Medial amygdaloid nucleus CAI-3: Field CAI, CA2 and CA3 of ammon' s horn in the hippocampus CG3: Cingulate cortex, area 3. DG: Dentate gyms (hippocampus) DMH: Dorsomedial hypothalamic nucleus DR: Dorsal raphe Fr2: Frontal cortex, area 2 LH: Lateral hypothalamic area MR: Medial raphe PAR 1: Parietal cortex, area I PVNm: Paraventricular hypothalamic nucleus, magnocellular region PVNp: Paraventricular hypothalamic nucleus, parvocellular region VMHC: Ventromedial hypothalamic nucleus, central region VMHDM: Ventromedial hypothalamic nucleus, dorsomedial region VMHVL: Ventromedial hypothalamic nucleus, ventrolateral region 103

G,, subunits in hypothalamus, midbrain and frontal cortex

The G protein a-subunits Gil• Gi2 , Gi3 and G0 were examined in the hypothalamus, midbrain and frontal cortex using immunoblots. Figure 13 presents

examples of Gi1, Gi2 and Gi3 proteins in the hypothalamus and midbrain. The levels of

Gi1 and Gi3 proteins were significantly decreased in the hypothalamus of rats that

received injections of fluoxetine for 7 and 14 days (Fig. 14A, 14C). The level of G0 proteins in hypothalamus was only significantly reduced in rats that received fluoxetine

injections for 7 days (Fig. 14D). The level of Gi2 proteins in the hypothalami of rats that received injections of fluoxetine was not significantly different from those that

received injections of saline (Fig 14B). The levels of G0 and Gi2 proteins in the midbrain were significantly decreased after 3 days of fluoxetine injections and remained below control levels for the duration of fluoxetine exposure (Fig. 15). The levels of

Gi1 and Gi3 proteins in the midbrain were not significantly reduced by injections of

fluoxetine (Fig. 15). Gi or G0 protein levels were not reduced in the frontal cortex of rats that received injections of fluoxetine (Table VI). 104

Hypothalamus

AS/7

Hypothalamus

AntiG;3

Mid brain

GC/2 -Ga

Veh 3 7 14 22 Days of fluoxetine (10 mg/kg . ip)

Fia=ure 13. Example of immunoblots of G proteins in brain regions from rats that received daily injections of fluoxetine. Top panel: Gi1 and Gi2 proteins in the hypothalamus; middle panel: Gi3 proteins in the hypothalamus; bottom panel: G0 proteins in the midbrain. The lanes from left to right contain membrane extracts from brain regions of rats that received: vehicle, fluoxetine for 3 days, fluoxetine for 7 days, fluoxetine for 14 days and fluoxetine for 22 days. 105

Hypothalamus B. Gi2 ...... ' ...... 0 0 I... I... 100 a. a. 1 20 CJ"l :::L 8 0 ~ 100

0 0 80 ""'0 60 ""'0 60 0 40 0 I... I...... 40 c c 0 20 0 u u 20

0 3 7 14 22 0 3 7 14 22 Days of fluoxetine Days of fluoxetine

::: C. Gi3 ...... 0 0 I... I... a. a. 1 00 '""" 100 cri cri ::t :::t 80 ,_ 80 * * ""'0 0 * 0 ""'0 60 60 '"""

0 40 0 40 ..... I... I...... c c 0 20 0 20 ..... u u

0 L-.CC::CL..J:CID1-.m:CIL..CCID1-.m:CIL-- 0 0 - 0 3 7 14 22 - 0 3 7 14 22 Days of fluoxetine Days of fluoxetine

Figure 14 (See next page for figure legend) 106

Figure 14. Fluoxetine reduces the levels of G proteins in the hypothalamus. The data represent mean ± S.E.M. of 6-8 rats per group. A.

Level ofGi1 proteins, One way ANOVA: F<4• 31>=3.197, P<0.05 B. Level ofGi2 proteins. One way ANOVA: F<4. 33>=0.4276, NS. C. Level of Gi3 proteins. One way ANOVA: F<4• 33> = 2.8079; P < 0.05. D. Level of G0 proteins. One way ANOVA: F<4• 35>=5.316, P<0.01. * Significant difference from the vehicle group, P <0.05 (Duncan's multiple range test) NS: No significant difference from the vehicle group. 107

Mid brain

-.... A. Gil ...... 0 0 \.... \.... a. 120 a. 100 O'l O'l 1 00 :::L :::t '-... 80 '-... * 0 0 80 0 0 60 ..._,, 60

0 40 \.... ~ 40 ...... c c 0 20 0 20 (.) () ....._ ...._ 0 0 0 0 3 7 14 22 0 .3 7 14 22 Days of fluoxetine Days of fluoxetine

100 .... * 80 .... * * 60 .... *

0 40 0 40 .... \.... I...... c c 0 20 0 20 ....

'+- 0 0 - 0 3 7 14 22 0 .3 7 14 22 Days of fluoxetine Days of fluoxetine

Figure 15 (See next page for figure legend) 108

Figure 15. Fluoxetine reduces the levels of G proteins in the midbrain.

The data represent mean ± S.E.M. of 6-8 rats per group. A. Level of Gi1 proteins, One way ANOVA: Fc4• 35>=0.9818, NS; B. Level of Gi2 proteins. One way

ANOVA: Fc4.31)=2.827, P=0.05 . C. Level of Gi3 proteins. One way ANOVA: Fc4• . 35> = 1.1669; NS . D. Level of G0 proteins. One way ANOVA: F<4 34>=6.56, P<0.01. *Significant difference from the vehicle group, P<0.05 (Newman Keuls' multiple range test). NS: No significant difference from the vehicle group. 109

TABLE VI

DAILY INJECTIONS OF FLUOXETINE DO NOT ALTER LEVELS OF G­ PROTEINS IN THE FRONTAL CORTEX

Saline 3 Days 7 Days 14 Days 22 Days

Gi1 93.1 ± 9.9 117.6±10.0 113.3±10.4 118.1±15.1 113.1±12.7

Gi2 102.4±10.6 99.5±10.5 123.7±21.1 94.7±11.1 128.1±26.3

Gi3 101.8± 5.5 120.8±16.0 157.5±28.1 133.9±22.6 133.9±26.0

Go 99.8± 6.4 99.6±12.0 85.9± 9.4 102.2± 9.4 110.7±18.7

The data represent means ± S.E.M. of the a subunits, expressed as % of the integrated optical density of the immunoblots/ µg protein. The data were obtained from 6-8 rats per group. 110 Discussion

The present results indicate that repeated injections of fluoxetine produce a

delayed and gradual desensitization of hypothalamic 5-HT1A receptors. This

desensitization is unlikely due to down-regulation of 5-HT1A receptors in the

hypothalamus or due to changes in the coupling of 5-HT1Areceptors to their G proteins.

The results of the present study demonstrate a similarity in time-course between the fluoxetine-induced reduction in hypothalamic levels of Gi proteins and the fluoxetine­ induced inhibition of neuroendocrine responses to a challenge with the 5-HT,A agonist

8-0H-DPAT. This similarity in time-courses suggests that hypothalamic Gil or Gi3

proteins may play a role in fluoxetine-induced desensitization of hypothalamic 5-HT1A receptors. The delay in desensitization of hypothalamic 5-HT tA receptors might underlie the delayed onset of therapeutic effects of fluoxetine and other 5-HT uptake inhibitors.

Hormone responses to 8-0H-DPAT

We used the changes in magnitude of elevation of plasma levels of ACTH, corticosterone and oxytocin, after injection of several doses of 8-0H-DPAT, as

indicators of desensitization of hypothalamic 5-HT1A receptors. 8-0H-DPAT increases plasma ACTH, corticosterone and oxytocin concentrations by activating hypothalamic

5-HT1A receptors. The ACTH and corticosterone responses to 8-0H-DPAT can be

inhibited by the 5-HT1A antagonists pindolol, spiperone, or WAY-100635 (Pan and

Gilbert, 1992; Gilbert et al.1988a; Critchley et al.1994a; Koenig et al.1987;

Przegalinski et al.1989) and the oxytocin responses to 8-0H-DPAT also can be 111 inhibited by NAN-190 and pindolol (Bagdy and Kalogeras, 1993). We have recently

observed that the 5-HT1A antagonist WAY-100635 (1 mg/kg, sc) blocks the effects of

8-0H-DPAT (50 µg/kg, sc) on plasma levels of ACTH, corticosterone and oxytocin

(Vicentic et al.1996). The location of the 5-HT1A receptors that stimulate the secretion of ACTH and oxytocin is likely in the hypothalamic paraventricular nucleus because injection of pindolol into the hypothalamic paraventricular nucleus inhibits the ACTH response to injection of 8-0H-DPAT into the same site (Pan and Gilbert, 1992).

Mechanical destruction of the hypothalamic paraventricular nucleus also inhibits the

ACTH, corticosterone and oxytocin responses to ipsapirone, a partial 5-HT1A agonist

(Bagdy and Makara, 1994). These data suggest that the ACTH and oxytocin responses

to 8-0H-DPAT are mediated by hypothalamic 5-HT1A receptors and can be used as markers for the functional status of hypothalamic 5-HT lA receptor systems.

The present observations of fluoxetine-induced desensitization of hypothalamic

5-HT1A receptors are consistent with our previous observations (Chapter IV), that daily injections of fluoxetine for 21 days produce a shift to the right in the dose-response effects of 8-0H-DPAT on plasma ACTH, corticosterone and oxytocin. In the present study, we further demonstrated that the desensitization of hypothalamic 5-HT IA receptors require at least 3 days of injections of fluoxetine. This delayed desensitization

of hypothalamic 5-HT1A receptors resembles the therapeutic delay seen in clinical studies and is different from the rapid desensitization (less than 24 hours) after a single

injection of 8-0H-DPAT and other 5-HT1A agonists (Kelder and Ross, 1992). The

difference between the SSRI's and the 5-HT1A agonists could be due to the fact that 5- 112

HT1A agonists can immediately and directly activate post-synaptic 5-HT1A receptors.

In contrast, 5-HT uptake inhibitors need to overcome the inhibitory influence of

somatodendritic 5-HT1A autoreceptors before they can induce an increase in the synaptic concentration of 5-HT in the hypothalamus, which in tum can activate post-synaptic 5-

HT1A receptors. Therefore, the present results support the hypothesis that

somatodendritic 5-HT1A receptors play a role in the delayed clinical improvement after onset of treatment with 5-HT uptake inhibitors.

A difference was observed between the effect of fluoxetine on ACTH and oxytocin responses to 8-0H-DPAT. ACTH showed no reduction in the maximal response to 8-0H-DPAT while oxytocin showed a reduction in the maximal responses to 8-0H-DPAT. This difference can be explained by the fact that a larger receptor reserve exists for the 5-HT1A receptor-mediated ACTH and corticosterone responses, than for the oxytocin response to 8-0H-DPAT (Meller and Bohmaker, 1994; Pinto et al.1994).

Autoradiogram of 3H-8-0H-DPAT binding

The present studies provide autoradiographic data on the distribution of 5-HT1A receptors in sub-regions of hypothalamic nuclei. While several studies have used autoradiographic analyses to examine other regions in the brain (Khawaja, 1995; Le

Poul et al.1995), few studies have examined the hypothalamus in the detail reported herein. In studies from other laboratories, only a few nuclei were examined (Frankfurt et al.1994; Hensler et al.1991; Pompeiano et al.1992; Palacios et al.1987). In the present study, several sub-divisions of hypothalamic nuclei were examined. The highest 113 density of 5-HT1A receptors in the hypothalamus was found in the ventromedial nucleus

(VMH). This observation is in agreement with studies using an antibody against the 5-

HT tA receptor, autoradiography or in situ hybridization for the mRNA coding for this receptor, (Wright et al.1995; Kia et al.1996; Frankfurt et al.1994; Hensler et al.1991;

Frankfurt et al.1993). Differences in the density of 5-HT1A receptors in the three sub­ divisions of the VMH (central, dorsomedial and ventrolateral) may be related to the

differences between males and females in the interaction between 5-HT1A receptors and gonadal steroids with respect to sexual behavior (Uphouse et al.1994a; Uphouse et al.1994b; Coirini et al.1992; Mendelson and Gorzalka, 1986). Physiological studies

indicate that activation of 5-HT1A receptors on neurons in the VMH activates their potassium conductance (Newberry, 1992).

The density of 5-HT1A receptors in the paraventricular nucleus (PVN) was lower than that in the surrounding tissue. The PVN can be roughly divided into parvocellular and magnocellular sub-regions. The parvocellular cells secrete corticotropin releasing hormone (CRH), which then stimulates the secretion of ACTH from the anterior pituitary gland. Serotonergic nerve terminals make direct synaptic contact with these

CRH cells (Liposits et al.1987). There are more serotonergic terminals innervating parvocellular CRH cells than magnocellular cells. Furthermore, oxytocin containing cells receive a higher innervation than vasopressin containing cells (Sawchenko et

al.1983). Parvocellular cells in the PVN contain a higher density of 5-HT1A receptors

than magnocellular cells which contain oxytocin and vasopressin. Activation of 5-HT1A receptors can stimulate the secretion of ACTH and oxytocin, but not vasopressin 114

(Bagdy and Kalogeras, 1993; Li et al.1993b; Van de Kar and Brownfield, 1993;

Vicentic et al.1996). Thus, the observation of a very low density of 5-HT1A receptors in the magnocellular cells in the lateral wing of the PVN is in agreement with the neuroendocrine data.

In addition to the hypothalamus, we determined the distribution of 5-HT1A receptors in nuclei of the amygdala, regions in the hippocampus, raphe nuclei and several cortical areas and layers. Neurons in the amygdala are involved in mood changes (Deakin, 1996; Handley, 1995; Adolphs et al.1994). Also, the central nucleus of the amygdala and hypothalamic paraventricular nucleus receive collateral projections from the dorsal raphe nucleus (Petrov et al.1994). It is possible that 5-HT uptake

inhibitors might also influence 5-HT1A receptors in the amygdala. Therefore, it was interesting to know whether repeated injections of fluoxetine would alter the density of

5-HT1A receptors and their coupling to G proteins. The hippocampus contains a very

high density of 5-HT1A receptors (Khawaja, 1995; Le Poul et al.1995). Several investigators studied the effects of long-term administration of 5-HT uptake inhibitors

on the hippocampal 5-HT1A receptors (Klimek et al.1994; Newman et al.1992; Hensler

et al.1991). With respect to the relative distribution of 5-HT1A receptors in hippocampal CAI, CA2 and CA3 regions, our results agree with those of other investigators (Khawaja, 1995). The cortex was also used as a standard for comparison because it has been studied extensively (Burnet et al.1994; Burnet et al.1995; Fanelli and Mcmonagle-Strucko, 1992). The cortex receives a different pattern of serotonergic inputs than the hypothalamus (Van Bockstaele et al.1993; Petrov et al.1992; Reichling 115 and Basbaum, 1991). This could mean that fluoxetine might not have the same effects on the cortex and hypothalamus. In the present studies, the cortex was used as a

control to test the region-selectivity of the effect of 5-HT uptake inhibitors on 5-HT1A

receptors and for comparison with other studies. The 5-HT1A receptors in the dorsal and median raphe nuclei are known as somatodendritic autoreceptors, involved in auto­ inhibition of the firing of serotonergic neurons. Neurons both in the dorsal and median raphe nuclei innervate the hypothalamus. Because changes in the release of 5-HT in

the hypothalamus are dependent on somatodendritic 5-HT1A autoreceptors, we included the dorsal and median raphe in the autoradiographic analysis of the distribution of 5-

HT tA receptors.

The present data suggest that fluoxetine-induced desensitization of 5-HT1A

receptors is not due to a reduction in the density of 5-HT1A receptors in the PVN. This is consistent with our previous results indicating that chronic injections of fluoxetine do

not alter the density or affinity of 5-HT1A receptors in whole hypothalamic homogenates

(Li et al.1994). Furthermore, the lack of changes in the density of 5-HT1A receptors in other brain regions is consistent with several investigations from other laboratories

(Le Poul et al.1995; Hensler et al.1991).

In the present study, we did not observe reductions in 3H-8-0H-DPAT labeled

5-HT1A receptors in either the dorsal raphe or median raphe nuclei. This result is consistent with other autoradiographic results indicating that chronic 5-HT uptake

inhibitors do not change the density of 5-HT1A receptors in the dorsal raphe and median rap he nuclei (Le Poul et al.1995; Hensler et al. 1991). 116

Autoradiographic analysis of Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding

The present studies provide the first autoradiographic data on Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding in the hypothalamus and other limbic brain regions. Gpp(NH)p inhibits the binding of 3H-8-0H-DPAT in most brain regions.

However, the degree of inhibition varies between different brain regions. This

variation in the degree of inhibition suggests that differential coupling of 5-HT1A receptors occurs in different brain regions.

3 8-0H-DPAT is a 5-HT1A agonist. One might expect that H-8-0H-DPAT, in the concentration of 2 nM, would mainly label the high affinity (coupled) state of 5-

HT1A receptors. Therefore, Gpp(NH)p should be able to inhibit most of the binding of

3 H-8-0H-DPAT to 5-HT1A receptors. However, results observed from the present and other studies indicate that 3H-8-0H-DPAT binding cannot be completely blocked by micromolar concentrations of Gpp(NH)p (Weinstein et al .1995). Butkerait et al. ( 1995) found no significant differences between the Bmax of 3H-8-0H-DPAT binding in Sf9

cells that express 5-HT1A receptors with G proteins and the same cells without G proteins. However, the Kd of 3H-8-0H-DPAT binding was significantly higher in the absence of G proteins (Kd = 7.2±0.9 nM) than in the presence of G proteins (Kd =

1.1 ± 0.2 nM). On the other hand, in many brain regions, the binding ratio of the 5-

3 3 HT1A agonist H-8-0H-DPAT to the antagonist H-WAY-100635 was more than 0.5, suggesting more than 50 % coupling of receptors (Gozlan et al.1995; Le Poul et al.1995; Kulikov et al.1995). This ratio is much higher than the ratio of agonist to

antagonist binding for other receptors. For example, the binding ratio of the 5-HT2A 12c 117

agonist DOI to the 5-HT2A antagonist ketanserin is about 0.2 (i.e. 20% of receptors are

coupled) (Teiteler et al.1990; Pinto and Battaglia, 1994). The high ratio of 3H-8-0H-

DPAT to 3H-WAY-100635 binding could be due to two possibilities: 1) a larger number

of 5-HT1A receptors are coupled to G proteins compared with most other receptors; or

2) 8-0H-DPAT binds to both the high and low affinity states of 5-HT1A receptors. The

present results and other studies support the latter possibility. This is possible because

the affinity of 8-0H-DPAT for G protein-coupled 5-HT1A receptors is close (less than

one logarithm scale) to that for uncoupled 5-HT1A receptors (Chamberlain et al.1993).

Taken together, these results suggest that 8-0H-DPAT may bind to both high and low

3 affinity states of 5-HT1A receptors. The degree of Gpp(NH)p-induced inhibition of H-

8-0H-DPAT binding might depend on the ratio of 5-HT1A receptors existing in the high

and low affinity states in each brain region.

Repeated injections of fluoxetine did not change the Gpp(NH)p-induced

inhibition of 3H-8-0H-DPAT binding. The lack of change suggests that no alteration

occurred in the fraction of coupled 5-HT1A receptors. This conclusion is consistent with the results reported by Le Poul et al. (1995) who observed that repeated injections of

fluoxetine did not change the ratio of 5-HT1A agonist (3H-8-0H-DPAT) to antagonist

(3H-W A Y-100635) binding.

Changes in the levels of Gi and G0 proteins induced by repeated injections of fluoxetine

The time-course of the effect of fluoxetine on Gi 1 and Gi3 proteins in the hypothalamus appears to parallel the reductions in ACTH and oxytocin responses to 8- 118

OH-DPAT, suggesting that the desensitization of 5-HT1A receptors may be due to decreased levels of Gil and Gi3 proteins. In vitro, both Gi1 and Gi3 proteins are known

to be coupled to 5-HT1A receptors (Raymond et al.1993). However, the hormone responses to 8-0H-DPAT were partly reduced after 3 days of fluoxetine injections,

while the hypothalamic levels of Gi1 and Gi3 proteins were not significantly reduced until 7 days. One explanation for this difference in time-course is that hormone

responses to 5-HT1A agonists are amplified through linkage via G proteins to the second messenger enzymes. Note that the hypothalamic levels of Gi1 and Gi3 proteins after 3 days of fluoxetine injections were 79.9 ± 6.8% and 82.6 ± 6.1 % respectively. After

7 days, the hormone responses to 8-0H-DPAT were almost maximally inhibited and the hypothalamic levels of Gil and Gi3 proteins were 58.0 ± 7.9% and 71.1 ± 10.43 respectively and statistically different from the control group. These observations suggest that a small change in the levels of G proteins might induce a large change in hormone responses.

The midbrain was examined for changes in G proteins because it contains serotonergic neurons in the dorsal and median raphe nuclei. The most salient finding is that the reduction in G proteins in the midbrain precedes the reductions of G proteins in the hypothalamus and that different G proteins are reduced in the midbrain than in

the hypothalamus. The levels of G0 and Gi2 proteins in the midbrain were reduced by repeated injections of fluoxetine at 3 days and remained low for the duration of the

injections. Since G0 proteins are coupled to 5-HT1A autoreceptors and probably play a role in the feedback inhibition of serotonergic firing (Sprouse and Aghajanian, 1988), 119 the decrease in their levels in the midbrain may be related to the desensitization of

somatodendritic 5-HT1A autoreceptors. This observation agrees with the hypothesis that

desensitization of the 5-HT1A autoreceptors in the raphe occurs before the

desensitization of postsynaptic 5-HT1A receptors. However, one cannot dismiss the

possibility that the reduction of G0 and Gi2 levels in the midbrain could also influence

the functions of other neurotransmitter receptors which are known to be coupled to G0 and Gi proteins.

Fluoxetine did not change the levels of Gi or G0 proteins in the frontal cortex at any time. This is consistent with the results reported by other investigators (Lesch and Manji, 1992; Lesch et al.1991a) and suggests that the regulation of the expression of these G proteins is region specific. The reason for the difference between brain regions is still unclear and deserves further investigation. It may be due to differences in the origin of serotonergic projections to frontal cortex versus the hypothalamus

(Mamounas et al.1991; Willoughby and Blessing, 1987), or to differences in co­ localized peptides in the serotonergic pathways that innervate the cortex and hypothalamus (Smith et al.1994; Li et al.1991; Francois-Bellan et al.1992), which could differentially modulate the regulation of G proteins. Other antidepressant drugs also differentially alter the expression of G proteins. For example, chronic injections of decrease the level of Gi proteins in the hypothalamus and the hippocampus but not in the frontal cortex (Lesch and Manji, 1992; Lesch et al.1991a). It is not clear

if the reduction in Gi and G0 proteins is due to reduced synthesis. Infusion of a lower

dose of fluoxetine (2.5 mg/kg/day) for 21 days did not alter Gi and G0 mRNA levels 120

in the hypothalamus and midbrain (Lesch and Manji, 1992; Lesch et al.1992b). This

could mean that fluoxetine does not reduce the synthesis of Gi and G0 proteins and that

the reduction is due to increased degradation or alterations in post-translational modification, such as reduced levels of palmitoylation of the G proteins (McCallum et

al.1995; Milligan et al.1995; Ross, 1995). On the other hand, this particular study

(Lesch and Manji, 1992; Lesch et al.1992b) used a lower dose of fluoxetine than the dose used in the present experiment and the lower dose might not have produced the

same reduction in the levels of G proteins observed in the present study.

It is difficult to reconcile the reduction in the levels of Gi1 and Gi3 in the hypothalamus with lack of change in the Gpp(NH)p-induced inhibition of 3H-8-0H­

DPAT binding. It should be noted that Gpp(NH)p-induced inhibition of 3H-8-0H­

DPAT binding is an indicator of a steady state condition of coupled 5-HT1A receptors.

Therefore, the Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding may not necessarily represent the dynamic status of agonist-induced coupling and signal

transduction of 5-HT1A receptors in living cells. In addition, it is possible that a decrease in the levels of G proteins will influence the coupling of G proteins to adenylyl cyclase, resulting in an alteration in the level of second messenger.

In summary, the results of the present study suggest that daily injections of fluoxetine produce a desensitization of 5-HT IA receptors in the hypothalamus. This desensitization occurs gradually and reaches a maximum after 14 days. The mechanism

of desensitization of hypothalamic 5-HT1A receptor systems may involve decreased levels of Gi 1 and/or Gi3 proteins. Furthermore, the earlier onset of reductions in 121

midbrain G0 proteins, compared with the decreases of hypothalamic Gi1 and Gi3 proteins, supports the hypothesis that sustained inhibition of 5-HT transporters first

desensitizes somatodendritic 5-HT1A autoreceptors in the raphe nuclei and, subsequently

desensitizes postsynaptic 5-HT1A receptors. CHAPTER VI

REPEATED INJECTIONS OF PAROXETINE PRODUCE A GRADUAL

DESENSITIZATION OF 5-HT1A RECEPTORS IN THE HYPOTHALAMUS

Summary

In the previous studies, we demonstrated that repeated injections of fluoxetine

produce a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus

of male rats. The desensitization of hypothalamic 5-HT1A receptors was evidenced by a reduction in hormone responses to 8-0H-DPAT and by reduction in hypothalamic

levels of Gil and Gi3 proteins which may be coupled to 5-HT1A receptors. However,

no changes were observed in the density of 5-HT1A receptors or in the coupling of 5-

HT IA receptors to their G proteins. The present study examined the time-course effects

of paroxetine on hypothalamic 5-HT1A receptors, to study whether the desensitization

of 5-HT1A receptors is mediated by the sustained blockade of 5-HT uptake sites. Male rats were injected with paroxetine (10 mg/kg, ip) once daily for 1, 3, 7 and 14 days.

Control rats received saline injections for 14 days. Eighteen hours after the last injection, the rats were challenged with 8-0H-DPAT (50 and 500 µg/kg, sc) and were decapitated 15 min after the injection of 8-0H-DPAT. Repeated injections of paroxetine reduced the 8-0H-DPAT-inducedelevation of plasma ACTH, corticosterone and oxytocin levels. The ACTH and corticosterone responses to the 50 µg/kg dose of

122 123 8-0H-DPAT, but not the high dose (500 µg/kg) of 8-0H-DPAT were significantly

reduced by repeated injections of paroxetine. On the other hand, the oxytocin

responses to both low and high doses of 8-0H-DPAT were reduced by the repeated

injections of paroxetine. ACTH and oxytocin responses to 8-0H-DPAT were

significantly reduced after 3 daily injections of paroxetine. The reduction reached a

maximum after 7 and 14 daily injections of paroxetine. The corticosterone response to 8-0H-DPAT was not significantly reduced until 7 days of paroxetine injections.

One daily injection of paroxetine did not alter the ACTH and corticosterone responses to 8-0H-DPAT. These results suggest that repeated injections of paroxetine produce

a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus. The

desensitization of hypothalamic 5-HT1A receptors require injections of paroxetine for more than one days.

Repeated injections of paroxetine did not alter the density of 5-HT1A receptors

in any brain region. However, the profile of changes in the levels of Gi and G0 proteins is somewhat different from that induced by fluoxetine. Injections of paroxetine

significantly reduced the levels of all four G proteins in the hypothalamus, although the

reduction in the levels of Gi1 and Gi3 proteins was greater than in Gi2 and G0 proteins.

The time-course of the reduction in the level of Gi3 proteins in the hypothalamus was

similar to the time-course of reduced hormone responses to 8-0H-DPAT. The

hypothalamic level of Gi1 proteins was significantly reduced after one day of paroxetine

injection and a reduced level of Gil proteins was maintained up to 14 days of injections.

Unlike fluoxetine, paroxetine significantly reduced the level of Gi2 proteins in the 124 hypothalamus after seven daily injections. The levels of all four G proteins in the

midbrain were decreased after the injections of paroxetine. Midbrain levels of Gi2 and

Gi3 proteins were significantly reduced after one daily injection of paroxetine and this reduced level was maintained until 14 days of injections. The level of Gil proteins was significantly decreased after three daily injections of paroxetine. Similar to fluoxetine,

paroxetine reduced the level of midbrain G0 proteins after three daily injections.

Although paroxetine did not alter the levels of Gi3 and G0 proteins in the frontal cortex, the levels of Gi1 and Gi2 proteins were significantly decreased in the frontal cortex after one day of paroxetine injection. These results suggest that repeated injections of

paroxetine reduce Gi and G0 proteins in a region-specific manner. Furthermore, the differential profiles of the effects of paroxetine and fluoxetine on the levels of G

proteins suggest that the reductions in the levels of Gi and G0 proteins are not only mediated by the blockade of 5-HT uptake sites, but also by side effects of these 5-HT uptake inhibitors. In conclusion, these results suggest that repeated injections of

paroxetine produce a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus. This desensitization is not due to changes in the density of hypothalamic

5-HT lA receptors. Since the time-course of the effect of paroxetine on the hypothalamic

level of Gi3 proteins is similar to the time-course of the hormone responses to 8-0H­

DPAT , the reduced Gi3 proteins may be partly involved in the desensitization of 5-

HT 1A receptors. In addition, the consistent effect of paroxetine and fluoxetine on the

level of G0 proteins in the midbrain suggests that a reduction of the level of G0 proteins

in the midbrain may play a role in the desensitization of 5-HT1A autoreceptors induced 125 by the repeated injection of 5-HT uptake inhibitors.

Introduction

5-HT uptake inhibitors, such as fluoxetine, paroxetine, fluvoxamine and sertraline are widely used to treat affective disorders (Wong et al.1995; Goldstein et al.1995; Weltzin et al.1994; Advokat and Kutlesic, 1995; Eriksson et al.1995; Lam et al.1995). The pharmacologic effect of these drugs is blockade of 5-HT uptake sites.

Because 5-HT uptake sites are responsible for reuptake of 5-HT from the synaptic cleft back into nerve terminals, blockade of 5-HT uptake sites will prolong the activity of 5-

HT and consequently increase the concentration of 5-HT in the synaptic cleft. Since the depletion of 5-HT has been hypothesized to be related to the pathology of depression, the blockade of 5-HT uptake sites by 5-HT uptake inhibitors should produce an immediate therapeutic effect on the treatment of depressed patients. However, the clinical application of these antidepressants suggests that the therapeutic effects of 5-HT uptake inhibitors may not be exclusively mediated by the blockade of 5-HT uptake sites because the clinical improvement is usually achieved 2-3 weeks after the onset of treatment. It is believed that the therapeutic effect of 5-HT uptake inhibitors may be related to adaptive changes induced by the blockade of 5-HT uptake sites.

Activation of 5-HT1A autoreceptors may be involved in the delay of the therapeutic effects of 5-HT uptake inhibitors. Recently, Studies (Artigas et al.1994;

Artigas, 1995; Blier and Bergeron, 1995) found that co-administration of the 5-HT1A/B adrenergic antagonist pindolol with 5-HT uptake inhibitors reduces the delay of clinical improvement in depressed patients. Our studies (Li et al.1996) (See Chapter V) have 126 demonstrated that repeated injections of fluoxetine produce a delayed and gradual

desensitization of hypothalamic 5-HT1A receptors. To determine whether the effect of

fluoxetine on the 5-HT1A receptors in the hypothalamus is mediated by blockade of 5-

HT uptake sites, another 5-HT uptake inhibitor paroxetine was used. Paroxetine has a chemical structure and pharmacokinetic profile that is different from fluoxetine

(Nemeroff, 1993; Van Harten, 1993; Goodnick, 1994). For example, paroxetine has a 10 fold higher affinity for 5-HT uptake sites than fluoxetine (Bolden-Watson and

Richelson, 1993; Richelson, 1994). Also, paroxetine has a higher ratio of affinity for serotonin/norepinephrine uptake sites than fluoxetine. The pharmacokinetics of paroxetine are quite different from those of fluoxetine (Goodnick, 1994; Van Harten,

1993; Richelson, 1994; Lane et al. 1995). In humans, the half-life of paroxetine (about

1 day) is much shorter than that of fluoxetine (3-5 days). Paroxetine does not have an active metabolite, while fluoxetine has an active metabolite, norfluoxetine, that has a long half-life (7-15 days). Also, one can expect that paroxetine will have different side effect than fluoxetine because of their different chemical structures. In fact, clinical application has shown that the side effects of paroxetine are different from those of fluoxetine (Lane et al.1995). However, since both fluoxetine and paroxetine inhibit 5-

HT uptake sites, a common effect of these two drugs would likely be mediated by blockade of 5-HT uptake sites. For example, there is no significant difference in their therapeutic effects for treatment of depression (Tignol, 1993; Lane et al.1995), suggesting that their therapeutic effect is mediated by blockade of 5-HT uptake sites.

5-HT1A receptors can be classified into somatodendritic and postsynaptic 127 receptors. Somatodendritic 5-HT1A autoreceptors are located on the 5-HT neurons in

the dorsal and median raphe nuclei in the midbrain. The function of the 5-HT1A autoreceptors is feedback regulation of 5-HT release from serotonergic nerve terminals.

Stimulation of 5-HT1A autoreceptors by 5-HT or 5-HT1A agonists will decrease the firing rate of neurons and subsequently reduce the release of 5-HT from nerve terminals.

Postsynaptic 5-HT1A receptors are distributed in most forebrain regions, such as the hippocampus, hypothalamus, amygdala and cortex (Kia et al.1996; Gozlan et al.1995;

Khawaja, 1995). The postsynaptic 5-HT1A receptors are activated by serotonergic inputs from 5-HT neurons and produce physiological responses, which depend on the

function of the target cells. For example, stimulation of 5-HT1A receptors in the hypothalamus will increase the secretion of several hormones, such as ACTH, corticosterone and oxytocin. Since plasma hormone concentrations are easy to measure,

the hormone responses to 5-HT1A agonists can be used as markers of the function of

hypothalamic 5-HT1A receptors. The magnitude of the hormone response to 5-HT1A

agonists reflects the function of 5-HT1A receptors in the hypothalamus.

5-HT1A receptors are G protein-coupled membrane proteins. So far, the

evidence suggests that 5-HT1A receptors are coupled to Gi or G0 proteins (Butkerait et

al.1995; Mulheron et al.1994; Fargin et al.1991). Somatodendritic 5-HT1A receptors

may be coupled to G0 proteins, which increase the opening of K+ channels and consequently suppress the activity of 5-HT neurons (Sprouse and Aghajanian, 1988;

Innis and Aghajanian, 1987; Innis et al.1988). Postsynaptic 5-HT1A receptors have a

higher affinity for Gi3 and Gi 1 proteins than that for Gi2 and G0 proteins (Raymond et 128 al.1993; Mulheron et al.1994; Bertin et al.1992). Stimulation of 5-HT1A receptors that are coupled to Gi proteins will inhibit the activity of adenylyl cyclase, resulting in a decrease of cAMP in the cells. In the present study, the effect of daily injections of

paroxetine on the levels of Gi and G0 proteins in the hypothalamus, midbrain and frontal cortex were determined to assess the effect of sustained inhibition of 5-HT uptake on the signal transduction system of 5-HT1A receptors.

We have hypothesized here that repeated injections of paroxetine would produce a delayed and gradual desensitization of 5-HT tA receptors in the hypothalamus. The time-course of the effect of paroxetine on the hormone responses to 8-0H-DPAT was

examined to assess the function of 5-HT1A receptors in the hypothalamus. To study

whether the desensitization is mediated by a down-regulation of 5-HT1A receptors, the

time-course of effect of paroxetine on the density of 5-HT1A receptors in the hypothalamus and several other brain regions was determined by autoradiographic analysis of 3H-8-0H-DPAT binding. Furthermore, we examined the levels of Gi and

G0 proteins in the hypothalamus, midbrain and frontal cortex, to determine whether a

reduction in the signal transduction system of 5-HT1A receptors plays a role in the

desensitization of 5-HT1A receptors.

Experimental Protocol

Male rats were injected with paroxetine (10 mg/kg, ip) once daily for 1, 3, 7 or 14 days. The control rats received saline injections for 14 days. Eighteen hours after the last injection, the rats were challenged with saline or 8-0H-DPAT (50 or 500

µg/kg, sc) and decapitated 15 min after the injection of 8-0H-DPAT. Trunk blood was 129 collected in centrifuge tubes containing 0.5 ml of 0.3M EDTA (pH 7.4) solution. After centrifugation at 2500 rpm, 4°C for 15 min, plasma aliquots were stored at -7D°C until they were used for hormone assays. The brains from saline challenged rats were quickly and carefully removed and frozen on powdered dry ice until the brains were completely frozen. The brains were then wrapped with plastic wrap, parafilm and aluminum foil and stored at -70°C until they were sectioned for autoradiography. The brains of the other rats were dissected and the hypothalamus, midbrain and frontal

cortex were stored at -70°C for immunoblots of Gi and G0 proteins.

Plasma ACTH, corticosterone and oxytocin were measured by

Radioimmunoassay as described in Chapter III. The density of 5-HT1A receptors in the hypothalamus and several other brain regions was analyzed by autoradiography (See

Chapter III for details). The levels of Gi1, Gi2, Gi3 and G0 proteins were analyzed using immunoblots as described in Chapter III.

Results

Hormone responses to 8-0H-DPAT

8-0H-DPAT significantly increased plasma ACTH concentration. Repeated injections of paroxetine significantly reduced the ACTH response to the 50 µglkg dose of 8-0H-DPAT (top panel of Fig.16). A partial reduction of the ACTH response to

8-0H-DPAT occurred after 3 daily injection of paroxetine and this inhibition was maximal after 7 and 14 days of injections. One daily injection of paroxetine did not alter the ACTH response to 8-0H-DPAT (bottom panel of Fig. 16). Repeated injections of paroxetine did not decrease the maximal effect of 8-0H-DPAT (500 µg/kg) 130 on ACTH secretion. However, the ACTH response to this high dose of 8-0H-DPAT

(500 µglkg) was potentiated by the daily injections of paroxetine for 7 days (top panel

of Fig. 16).

Figure 17 shows the effect of both 8-0H-DPAT doses (top) and a time-course

(bottom) of the effect of repeated injections of paroxetine on the corticosterone response to 8-0H-DPAT. 8-0H-DPAT significantly increased plasma corticosterone

concentration. Repeated injections of paroxetine significantly inhibited the corticosterone response to the 50 µglkg dose of 8-0H-DPAT. Unlike the ACTH response, the reduced corticosterone response to 8-0H-DPAT was statistically

significant only after 7 daily injections of paroxetine (bottom panel of Fig.17).

Injections of paroxetine for one or three days did not decrease the corticosterone response to 8-0H-DPAT. However, the corticosterone response to the high dose (500

µglkg) of 8-0H-DPAT was significantly potentiated by repeated injections of paroxetine for three days (top panel of Fig. 17).

As is shown in Figure 18, 8-0H-DPAT significantly increased plasma oxytocin concentration. Paroxetine significantly decreased the oxytocin response to both low and high doses of 8-0H-DPAT after 3 daily injections. The reduction of the oxytocin response to 8-0H-DPAT reached a maximum after 7 and 14 daily paroxetine injections

(bottom panel of Fig. 18). Injection of paroxetine for 1 day did not reduce the effect of 8-0H-DPAT on oxytocin. However, the oxytocin response to the high dose (500

µglkg) of 8-0H-DPAT was significantly reduced by injection of paroxetine for one day

(top panel of Fig. 18). 131

e SALINE .& PAROXETINE 1 DAYS ..... PAROXETINE 3 DAYS + PAROXETINE 7 DAYS ,.,....-._ • PAROXETINE 14 DAYS E 1200 '-..., 01 1000 a. '-"" 800 * I I- 600 u <( 400 <( ~ 200 (11 <( ~ _J 0 - a_ 0 50 500 Dose of 8-0H-DPAT (µ.g/kg sc)

c:::=i SALi NE ,,-.... ~ 8-0H-DPAT (50 µ.g/kg sc) E 800 * '-..., Ol * ....__,a. 600

I I- u 400 <( <( t :2 200 t (11 <( _J a_ 0 VHE 7 14 Days of paroxetine

Fi2ure 16. Daily injections of paroxetine inhibit the effect of 8-0H-DPAT on plasma ACTH. Top: low and high doses of 8-0H-DPAT; Bottom: time-course of the reduction observed with an 8-0H-DPAT dose of 50 µglkg sc. The data represent mean ± S.E.M. of 8-12 rats per group. Two way ANOVA: Main effect of paroxetine: Fc4• 125l=4.945 P<0.001; Main effect of 8-0H-DPAT: Fcz. 125)=108.013 P< 0.001; Interaction betweenparoxetine and 8-0H-DPAT: Fcs. 125)=9.1176 P <0.001. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, P<0.05; + Significant difference from the saline (0 dose of paroxetine) group, P < 0.05 (Newman Keuls' multiple range test) 132

e SALINE £ PAROXETINE 1 DAYS T PAROXETINE 3 DAYS + PAROXETINE 7 DAYS Q) .30 • PAROXETINE 14 DAYS c ... 0 I... Q) 25 +-' (/} ...... 20 * ------7.:_~~~: l 0 "O ...... £·-- ,,,...... ······ ' (.) ,.,..,,. .,,,"" ···········" ...... ""-... 15 / .L .... * I... CJ1 r' ,,,,.""' ········· / ...... 10 / ...... 3 3 / .... .- """""'•••• ····· .L 0 E 5 t rn 0 0 a.. 0 50 500 Dose of 8-0H -DPAT (µg/kg sc)

C=:J SALINE

Q) ~ 8-0H-DPAT c * (50 µg/kg sc) 0 20 I... * ....CV en ...--.. 1 5 0 "'O () t ~ ~ 10 0 :::t (.) ...... _,

0 5 E (/) 0 0 CL VHE 3 7 14 Days of paroxetine

Fia=ure 17. Daily injections of paroxetine inhibit the effect of 8-0H-DPAT on plasma corticosterone. Top: low and high doses of 8-0H-DPAT; Bottom: time­ course of the reduction observed with an 8-0H-DPAT dose of 50 µg/kg sc. The data represent mean ± S.E.M. of 8-12 rats per group. Two way ANOVA: Main effect of paroxetine: F<4. 123)=2.9577 P<0.001; Main effect of 8-0H-DPAT: F<2• 123)=77.1356 P<0.001; Interaction between paroxetine and 8-0H-DPAT: F<8. 123)=2.653 P<0.05. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, P<0.05; + Significant difference from the saline (0 dose of paroxetine) group, P<0.05 (Newman Keuls' multiple range test) 133

e SALINE .t.. PAROXETINE 1 DAYS Y PAROXETINE 3 DAYS + PAROXETINE 7 DAYS E • PAROXETINE 14 DAYS ""'- 120 * O'I • ~ 100 c 80 (.) .8 60 >. x 40 0 0 20 E en 0 0 a.. 0 50 500 Dose of 8-0H -DPAT (µ.g/kg sc)

c=J SALINE E * ~ 8-0H-DPAT ci; 50 (50 µg/kg sc} a.. ...__,, 40 c

·-(J 30 ...... 0 ~ 20 0 0 1 0 t Cll 0 0 a... VHE 3 7 14 Days of paroxetine

Figure 18. Daily injections of paroxetine inhibit the effect of 8-0H-DPAT on plasma oxytocin. Top: low and high doses of 8-0H-DPAT; Bottom: time-course of the reduction observed with an 8-0H-DPAT dose of 50 µg/kg sc. The data represent mean ± S.E.M. of 8-12 rats per group. Two way ANOVA: Main effect of

paroxetine: Fc4• 122>=12.8076 P<0.001; Main effect of 8-0H-DPAT: Fc2, 122)=401.5 P<0.001; Interaction between paroxetine and 8-0H-DPAT: Fc8, 122)=3.82 P<0.01. *Significant difference from the saline (0 dose of 8-0H-DPAT) group, P<0.05; + Significant difference from the saline (0 dose of paroxetine) group, P < 0.05 (Newman Keuls' multiple range test). 134 Autoradiographic analysis of 3H-8-0H-DPAT binding

Autoradiographic analysis of 3H-8-0H-DPAT binding was used to examine the

effect of paroxetine on the density of 5-HT1A receptors. The time course of the effect

of repeated injections of paroxetine on the density of 5-HT1A receptors was determined

in several nuclei in the hypothalamus, amygdala, hippocampus and dorsal and median

raphe nuclei as well as several layers of the cortex. As shown in Table VII, the

distribution of 5-HT1A receptors varies among brain regions, which is consistent with

that seen in the previous study (Chapter V). Like fluoxetine, paroxetine did not alter

the density of 5-HT1A receptors in any brain regions after daily injections (Table VII).

Levels of Gi and G0 proteins in the hypothalamus, midbrain and frontal cortex

The effects of repeated injections of paroxetine on the levels of Gi 1, Gi2 , Gi3 and

G0 proteins were determined using immunoblots. The four G proteins were differentially influenced in the hypothalamus, midbrain and frontal cortex by repeated

injections of paroxetine.

Repeated injections of paroxetine significantly reduced the levels of all four G proteins in the hypothalamus, although the reduction in the levels of Gil and Gi3 proteins

was greater than that of Gi2 and G0 proteins (Fig. 19). The level of Gu proteins was

significantly reduced after one day of paroxetine injection and the reduced level of Gi1

proteins was maintained until 14 days of injections (Fig.19A). Paroxetine significantly

decreased the level of Gi3 and G0 proteins after 3 days of injections (Fig. 19C, D). The

low level of Gi3 proteins was maintained until 14 daily injections of paroxetine (Fig.

19C), while the level of G0 proteins returned to normal levels after 14 days of injections 135

(Fig. 19D). The time-course of the effect of paroxetine on the level of Gi3 protein was similar to the time-course in reducing the hormone responses to 8-0H-DPAT. The level of Gi2 proteins was significantly decreased only after 7 daily paroxetine injections

(Fig. 19B).

As shown in Fig. 20, the levels of all four G protein were decreased in the

midbrain after daily injections of paroxetine. The levels of Gi2 and Gi3 proteins were significantly reduced after one day of injection of paroxetine and the reduced level was maintained until 14 days of injections (Fig. 20B, C). The level of Gn proteins was significantly decreased after 3 days of injections of paroxetine (Fig. 20A). Paroxetine

reduced the level of G0 proteins in the midbrain after 3 and 7 daily injections (Fig,

20D).

Although paroxetine did not alter the levels of Gi3 and G0 proteins in the frontal

cortex, the levels of Gi1 and Gi2 proteins were significantly decreased in the frontal cortex after injection of paroxetine for one day (Fig. 21A,B). 136 TABLE VII

REPEATED INJECTIONS OF PAROXETINE DO NOT ALTER THE DENSITY

OF 5-HT1A RECEPTORS AT ANY BRAIN REGION

3 Brain regions Nuclei Density of H-8-0H-DPAT - labeled 5-HT1A receptors Saline 1 days 3 days 7 days 14 days Cortex Fr2 layer 1-3 26.5±1.5 22.9±1.7 23.2±1.6 23.3±1.1 22.9±2.2 layer 5 72.6±3.6 64.1 ±2.6 64.6±3.4 62.1 ±3.2 64.2±3.9 ------~l!X~~-~- §l._l_t_~:.~-.?~·Qt_~:.~_.?.7-:±t_'!:.~_.?_~._Q_;!;.~:.~-~~.:~.;t~..:l_ CG3 layer 1-3 44.9±4.4 40.3±2.2 41.4±4.3 40.4±3.3 37.8±3.8 layer 5 65.5±5.6 54.9±3.5 56.7±4.4 57.9±4.3 56.3±5.1 layer 6 62.5±4.5 51.6±1.8 52.1±3.3 55.0±3.8 54.8±4.6 Hypothalamus PVNm 7.0±0.6 5.8±0.4 5.6±0.3 6.1 ±0.7 6.6±0.7 PVNp 8.3±0.7 7.3±0.4 7.2±0.4 8.0±0.7 8.6±0.6 AHN 12.8±0.6 10.8±0.5 10.6±1.0 10.9±1.2 12.3±0.8 LH 4.7±0.3 4.2±0.3 4.2±0.4 4.3±0.2 4.7±0.2 VMHDM 21.6± 1.6 21.8± 1.4 21.2± 1.1 19.4±1.0 18.9± 1.0 VMHC 32.8±2.6 32.2±2.4 31.2±2.1 27.8±1.9 29.1±1.8 VMHVL 19.8±1.3 19.2±0.9 19.2±1.1 17.7 ±1.0 17.1 ±0.8 DMH 10.0±0.9 9.8±0.7 10.0±0.4 8.6±0.3 9.1±0.3 Amygdala AC 6.1 ±0.4 6.5 ±0.5 6.4±0.2 6.1 ±0.3 5.4±0.3 ALP 11.5±0.7 11.1±0.7 13.1±0.4 11.8±0.7 11.2±0.4 ABL 17.2±1.1 16.7±0.8 17.3±0.7 17.2±0.7 16.8±0.6 ABM 26.5±2.1 24.3±2.2 24.5±1.7 23.0±1.1 24.1±1.3 AM 18.7±2.1 18.1±1.0 18.6±0.7 18.0±0.9 17.8±0.6 Hippocampus CAl 62.9±1.7 49.1±5.8 51.9±3.0 43.9±4.7 59.0±4.0 CA2 14.5±0.4 12.2±1.3 11.6±0.5 10.6±0.9 13.6±0.8 CA3 31.6±1.1 26.5±3.2 27.3±2.0 25.2±3.2 31.9±2.3 DG 84.1+3.0 66.5±5.5 70.7±4.4 63.2±6.4 78.3±5.2 Midbrain DR 30.8±1.8 32.4±1.5 32.0±0.9 32.9±0.4 31.9±0.9 MR 21.7+1.7 23.4+2.2 20.5± 1.2 20.2±1.4 18.5±0.9 137

The data represent mean ± S.E.M. of 5-6 rats per group.

Abbreviations: ABL: Basolateral amygdaloid nucleus ABM: Basomedial amygdaloid nucleus AC: Central amygdaloid nucleus AHN: Anterior hypothalamic nucleus ALP: Lateral amygdaloid nucleus, posterior region AM: Medial amygdaloid nucleus CAl-3: Field CAl, CA2 and CA3 of ammon's horn in the hippocampus CG3: Cingulate cortex, area 3. DG: Dentate gyrus (hippocampus) DMH: Dorsomedial hypothalamic nucleus DR: Dorsal raphe Fr2: Frontal cortex, area 2 LH: Lateral hypothalamic area MR: Medial raphe PAR 1: Parietal cortex, area 1 PVNm: Paraventricular hypothalamic nucleus, magnocellular region PVNp: Paraventricular hypothalamic nucleus, parvocellular region VMHC: Ventromedial hypothalamic nucleus, central region VMHDM: Ventromedial hypothalamic nucleus, dorsomedial region VMHVL: Ventromedial hypothalamic nucleus, ventrolateral region 138

Hypothalamus

,,.-....

+-' +-' 0 0 L. L. a. 1 00 a. 100 CJ1 :::l 80 80 0 '0 60 60

0 40 0 40 !.... L. -'-' +-' c g 20 0 20 (j (J

0 0 0 3 7 14 - 0 3 7 14 Days of paroxetine Days of paroxetine

Gi3 :- C. -'-' 0 0 !.... !.... a.. 100 a.. 1 00 - CJ'l :::l 80 80 - '0 * * 0 60 60 -

0 40 0 40 L- L. L. -'-' -'-' c c 0 20 0 20 .... u (.)

"'-- 0 L-J:IIIIL..CIJCID....EIIIIL...cc:m...... JCIJIIIL-- 0 0 3 7 1 4 0 3 7 14 Days of paroxetine Days of paroxetine

Figure 19 (see next page for figure legend) 139

Fi~ure 19. Paroxetine reduces the levels of G proteins in the hypothalamus.

The data represent mean ± S.E.M. of 6-8 rats per group. A. Level of Gi1 proteins,

One way ANOVA: F<4• 24) =3.7267, P<0.05; B. Level of Gi2 proteins. One way

ANOVA: F<4• 25)= 1.892, NS. C. Level of Gi3 proteins. One way ANOVA: F<4.

27)=2.8918, P<0.05; D. Level of G0 proteins. One way ANOVA: F<4• 28) =3.3027, P<0.05. * Significant difference from the vehicle group, P < 0.05 ( Newman Keuls' multiple range test) 140

Mid brain . +-' +-' 0 0 L L a... 1 00 a... 100 Ol Ol :::t 80 ::3... 80 ...... 0 0 0 60 0 60

0 40 0 40 L L +-' c c 0 20 0 20 () ()

0 0 0 3 7 14 - 0 3 7 14 Days of paroxetine Days of paroxetine

+-' +-' 0 0 L ~ 100 .... a.. 1 00 O" Ol 80 .... * :::t 8 0 :::t ...... 0 0 0 60 ...... __,0 60 *

0 40 0 40 .... L L +-' +-' c c 0 20 0 20 .... () ()

0 0 - - 3 7 14 0 3 7 14 0 ~ Days of paroxetine Days of paroxetine

Figure 20 (see next page for figure legend) 141

Fi2ure 20. Paroxetine reduces the levels of G proteins in the midbrain. The

data represent mean ± S.E.M. of 6-8 rats per group. A. Level of Gi 1 proteins, One way ANOVA: F<4• 24l =19.55, P<0.01 B. Level of Gi2 proteins. One way ANOVA:

F<4• 24l =12.6; P<0.01 . C. Level of Gi3 proteins. One way ANOVA: F<4• 24l=8.809,

P<0.01. D. Level of G0 proteins. One way ANOVA: F<4• 27l=4.449; P<0.01 * Significant difference from the vehicle group, P < 0.05 (Newman Keuls' multiple range test) 142

Frontal cortex

+-' ...... 0 0 .._ 1 00 \... a.. a.. 1 00 CTI CTI ::t 80 :::t 8 0 '--.. '--.. Cl Cl 0 60 0 60 ....._,,

0 40 0 40 \... \... +-' +-' c c 0 20 0 20 (.) (.)

0 0 0 0 - 0 3 7 14 - 0 3 7 14 Days of paroxetine Days of paroxetine

-: C. Gi3 +-' 0 0 I... 1 00 I... Q.. a.. 1 00 .... CJ'I ::3.. 80 '--.. 80 - 0 0 60 60 -

0 40 0 40 '- .._ I...... c c 0 20 0 20 '- u 0 -0 0 3 7 14 0 3 7 14 ~ Days of paroxetine Days of paroxetine

Figure 21 (see next page for figure legend) 143

Fi2Ure 21. Paroxetine reduces the levels of G proteins in the frontal cortex.

The data represent mean ± S.E.M. of 6-8 rats per group. A. Level of Gi 1 proteins, One way ANOVA: F<4. 30)=12.045; P<0.01 B. Level of Gi2 proteins. One way ANOVA: F<4• 3l=5.189, P=0.01 . C. Level of Gi3 proteins. One way ANOVA: F<4•

32l=0.9768, NS. D. Level of G0 proteins. One way ANOVA: Fc4. 28l=0.289, NS. * Significant difference from the vehicle group, P<0.05 (Newman Keuls' multiple range test) 144 Discussion

The results of the present study agree with the data obtained with fluoxetine and

suggest that sustained blockade of 5-HT uptake produces a delayed and gradual

desensitization of 5-HT1A receptors in the hypothalamus. This desensitization of 5-HT1A

receptors is unlikely due to a down-regulation of hypothalamic 5-HT IA receptors. Since

the time-course of paroxetine-induced reduction in the level of Gi3 proteins in the

hypothalamus is similar to the time-course of reduction in the hormone responses to 8-

0H-DPAT, it is possible that the reduction in the level of Gi3 proteins is involved in

the desensitization of hypothalamic 5-HT1A receptors.

In the present study, we examined changes in 5-HT1A receptors after 1, 3, 7 and

14 daily injections of paroxetine. In contrast, the study on the effects of fluoxetine had

a time-course of 0, 3, 7, 14 and 22 days. The reason for adding a one day paroxetine

group was to determine when the desensitization of the hypothalamic 5-HT1A receptors would starts. We have observed that 3 daily injections of fluoxetine partially reduce the hormone responses to 8-0H-DPAT. In the present study, we determined whether

the desensitization of hypothalamic 5-HT1A receptors occurs after one daily injection of paroxetine. We deleted the group of 22 daily injections in the present study because

the maximal effects of fluoxetine on the 5-HT1A receptors were observed after 14 daily injections. Comparing the time-course of fluoxetine with that of paroxetine, these

results suggest that the desensitization of 5-HT1A receptors in the hypothalamus requires more than one day of sustained inhibition of 5-HT uptake. However, one daily

injection of paroxetine significantly decreases the levels of Gi1 proteins in the 145 hypothalamus, the levels of Gi2 and Gi3 proteins in the midbrain and the levels of Gil and Gi2 proteins in the frontal cortex. These results suggest that paroxetine induces some changes after one daily injection. These changes may not be related to the

function of hypothalamic 5-HT1A receptors, because the neuroendocrine response is an

amplified response to activation of the hypothalamic 5-HT1A receptors and it was not reduced after one daily injection of paroxetine.

In the present study, ACTH, corticosterone and oxytocin responses to 8-0H­

DPAT were used to assess the function of hypothalamic 5-HT1A receptors. Several studies have demonstrated that 8-0H-DPAT-induced increases of plasma ACTH, corticosterone and oxytocin are dose-dependent. The ACTH and corticosterone responses to 8-0H-DPAT can be abolished by the 5-HTIA antagonists, pindolol, spiperone, NAN-190, UH-301, WAY-100135 and WAY-100635 (Cowen et al.1990;

Lejeune et al.1993; Pan and Gilbert, 1992; Critchley et al.1994b; Przegalinski et al.1989; Kelder and Ross, 1992; Vicentic et al.1996). The oxytocin response to 8-0H­

DPAT can be inhibited by WAY-100635 and NAN-190 (Vicentic et al.1996; Bagdy and

Kalogeras, 1993). A lesion in the hypothalamic paraventricular nucleus blunted the

ACTH, corticosterone and oxytocin responses to ipsapirone (Bagdy and Makara, 1994;

Bagdy, 1995). These data suggest that the hormone responses to 8-0H-DPAT are

mediated by activation of 5-HT1A receptors in the hypothalamus, likely in the paraventricular nucleus. Therefore, the magnitude of hormone responses to 8-0H­

DPAT can be used as a tool to assess the function of hypothalamic 5-HT1A receptors

(Van de Kar, 1991; Van de Kar, 1989; Van de Kar and Brownfield, 1993). The results 146 of the present study show that repeated injections of paroxetine significantly decrease the hormone response to 8-0H-DPAT, suggesting that paroxetine produces a

desensitization of 5-HT1A receptors in the hypothalamus. The desensitization of

hypothalamic 5-HT1A receptors appears after 3 daily injections and reaches a maximum effect after 7 and 14 days. These results are consistent with those observed with

repeated injections of fluoxetine, suggesting that the desensitization of 5-HT1A receptors is mediated by the blockade of 5-HT uptake sites.

It was unexpected that the ACTH and corticosterone responses to the 500 µg/kg dose of 8-0H-DPAT were significantly potentiated by 3 or 7 daily injections of paroxetine. However, at the same time points, the ACTH and corticosterone responses to the low dose (50 µg/kg) of 8-0H-DPAT were decreased by daily injections of paroxetine. The 50 µg/kg dose of 8-0H-DPAT is close to the ED50 dose for ACTH

and corticosterone and is more sensitive to changes in the function of 5-HT1A receptors.

On the other hand, the 500 µg/kg dose of 8-0H-DPAT is a maximal dose for ACTH and corticosterone. At this high dose, 8-0H-DPAT may stimulate some other receptors for which 8-0H-DPAT has a relative high affinity. For example, the affinity of 8-0H­

DPAT for 5-HT7 receptors is about 10-fold lower than that for 5-HT1A receptors (Hoyer et al.1994). In addition, no potentiation of the effects of 8-0H-DPAT was observed in the oxytocin response. All together, the potentiation of the AC TH and corticosterone responses to the high dose of 8-0H-DPAT may not be mediated by hypothalamic 5-

HT IA receptors.

Similar to our previous observations with fluoxetine, daily injections of 147 paroxetine decreased the ACTH and corticosterone responses to a low dose, but not a high dose of 8-0H-DPAT, while the decrease in the oxytocin response was observed

at both low and high doses of 8-0H-DPAT. There is a higher 5-HT1A receptor reserve

for ACTH and corticosterone responses than for the oxytocin response to 5-HT1A agonists (1994; 1994), unpublished data from Pinto and Battaglia). This difference in receptor reserve might explain the difference between ACTH and oxytocin. However, it is also possible that the lack of change in the ACTH and corticosterone responses to a high dose of 8-0H-DPAT is due to activation of other receptors, which increase

ACTH and corticosterone secretion and may be sensitized by repeated injections of paroxetine.

Repeated injections of paroxetine did not alter the density of 5-HT1A receptors in any brain regions. This observation is in agreement with our observations with fluoxetine and also in agreement with the data reported by other investigators (Le Poul et al.1995; Hensler et al.1991). Le Poul et al. (1995) showed that neither fluoxetine

nor paroxetine altered the density of 5-HT1A receptors in the dorsal raphe nucleus and in the dentate gyms of the hippocampus. Hensler et al. ( 1991) found that repeated

injections of sertraline or citalopram did not influence the density of 5-HT1A receptors in several brain regions.

There are similarities but also differences between paroxetine and fluoxetine with respect to their effects on the levels of G proteins. Overall. paroxetine seems to produce a greater and earlier reduction in the levels of Gi and G() proteins, especially

the levels of Gi1 and Gi2 proteins. For example, paroxetine reduced the level of Gi1 148 proteins in the hypothalamus after one daily injection, while fluoxetine did not

significantly reduce the level of Gi 1 proteins until 7 daily injections. Similarly, fluoxetine did not significantly alter the hypothalamic level of Gi2 proteins, but paroxetine decreased the level of Gi2 proteins after 7 daily injections. So far, there is

no good explanation for the differential influence on the levels of Gi and G0 proteins by fluoxetine and paroxetine, since little is known regarding the mechanism though which they decrease G protein levels.

Beside the differential effect of fluoxetine and paroxetine on levels of G proteins, there are some similarities which may be more important for the

desensitization of 5-HT1A receptors. For example, the time-course of reduction in the hypothalamic level of Gi3 proteins is similar to the time-course of hormone responses to 8-0H-DPAT after repeated injections of paroxetine. This observation is consistent

with the results observed with repeated injections of fluoxetine. Since 5-HT1A receptors have a high affinity for Gi3 proteins, this similarity in time-courses for reduction in the hypothalamic level of Gi3 proteins and the neuroendocrine resporu;es to 8-0H-DPAT suggests that the reduction in the level of Gi3 may be involved in the desensitization of

hypothalamic 5-HT1A receptors.

Repeated injections of paroxetine significantly decreased the level of Gi2 proteins in the midbrain. The reduction in the level of Gi2 proteins appeared after one daily

injection and remained for 14 daily injections. Paroxetine reduced the level of G0 proteins after 3 daily injections. These changes are similar to the results observed with

fluoxetine injections. The level of G0 proteins returned to normal after 14 daily 149

injections of paroxetine, which is in contrast with fluoxetine-induced reduction of G0 proteins which remained reduced during the 22 daily injections. Desensitization of 5-

HT1A autoreceptors in the dorsal raphe nucleus has been detected after 3 to 14 daily injections of fluoxetine or paroxetine (Le Poul et al.1995). However, no data are available on the effect of one daily injection of 5-HT uptake inhibitors on 5-HT1A autoreceptors. Therefore, it is difficult to determine which G proteins are involved in the desensitization of 5-HT1A autoreceptors.

In conclusion, the results of the present of study suggest that repeated injections of paroxetine produce a delayed and gradual desensitization of hypothalamic 5-HT1A receptors. This desensitization is similar to that induced by fiuoxetine, suggesting that sustained blockade of 5-HT uptake sites mediates this effect. Since no change was detected in the density of 5-HT1Areceptors in the hypothalamic nuclei, it is unlikely that the desensitization of hypothalamic 5-HT1Areceptors is due to a down-regulation of 5-

HT1A receptors. The similarity of the time-course of the leYel of Gi3 proteins to the time-course of the hormone responses to 8-0H-DPAT, after repeated injections of paroxetine, suggests that the reduction of the level of Gll proteins may be partly involved in the desensitization of 5-HT1A receptors. CHAPTER VII

GENERAL DISCUSSION

The present studies demonstrate that prolonged blockade of 5-HT uptake sites by 5-HT uptake inhibitors produces a delayed and gradual desensitization of

hypothalamic 5-HT1A receptors. This desensitization is not induced by blockade of norepinephrine uptake sites following long-term administration of desipramine. Because

neither fluoxetine nor paroxetine changes the density and affinity of 5-HT1A receptors

in the hypothalamus, the desensitization of hypothalamic 5-HT1A receptors is unlikely due to receptor down-regulation. A reduction in the level of Gi3 proteins may play a

role in the desensitization of hypothalamic 5-HT1A receptors, because the time-course of the reduction in the level of Gi3 proteins is similar to the time-course of the reduction of the hormone responses to 8-0H-DPAT. Therefore, the results of the present studies support our hypothesis that repeated injections of 5-HT uptake inhibitors produce a

delayed and gradual desensitization of hypothalamic 5-HT1A receptors that may be due to changes in their signal transduction mechanisms.

The Neuroendocrine Challen1:e Test --- A Tool to Examine the Function of

Hypothalamic 5-HT1A Receptors

In the present studies, ACTH, corticosterone and oxytocin responses to the 5-

HT1A agonists 8-0H-DPAT and ipsapirone were used to eyaluate the function of

150 151 hypothalamic 5-HT1A receptors. Several studies have demonstrated that ACTH,

corticosterone and oxytocin responses to 5-HT1A agonists are mediated by 5-HT1A

receptors (Van de Kar, 1991; Van de Kar and Brownfield, 1993). First, several 5-HT1A agonists, such as 8-0H-DPAT, ipsapirone, buspirone and gepirone stimulate the secretion of ACTH, corticosterone and oxytocin in a dose-dependent manner (Cowen et al.1990; Fuller, 1992; Gilbert et al.1988a; Koenig et al.1988; Bagdy and Kalogeras,

1993). Second, the hormone responses to 5-HT1A agonists can be inhibited by 5-HT1A antagonists, such as pindolol, spiperone, NAN-190, UH-301, WAY-100135 and WAY-

100635 (Cowen et al.1990; Lejeune et al.1993; Pan and Gilbert, 1992; Critchley et al.1994b; Przegalinski et al.1989; Kelder and Ross, 1992; Bagdy and Kalogeras, 1993;

Vicentic et al.1996), but not by 5-HTrn, 5-HT2 , 5-HT3 and adrenoceptor antagonists

(Przegalinski et al.1989). Third, the hormone responses to 5-HT1A agonists may be

mediated by postsynaptic 5-HT1A receptors. A lesion in 5-HT neurons by intracerebroventricular injection of 5,7-DHT or depletion of 5-HT with PCPA did not

decrease the effects of 5-HT1A agonists on the secretion of ACTH and corticosterone

(Przegalinski et al.1989; Gilbert et al.1988b and our unpublished data). Furthermore, a lesion in the paraventricular nucleus of the hypothalamus blunted the corticosterone and oxytocin responses to ipsapirone (Bagdy and Makara, 1994; Bagdy, 1994).

Together, these results suggest that 5-HT1A agonists increase the section of ACTH, corticosterone and oxytocin by activating postsynaptic 5-HT tA receptors in the

hypothalamus. Therefore, the magnitude of the hormone responses to 5-HT1A agonists

can be used as an index for the function of hypothalamic 5-HT 1A receptors. 152

Although it is clear that stimulation of 5-HT1A receptors in the hypothalamus increases the secretion of ACTH, corticosterone and oxytocin, the intracellular pathway

of the 5-HT1A receptor-mediated regulation of hormone secretion is still unknown. For example, no data are available regarding which G proteins and second messengers mediate the increase in the secretion of hormones, and how the second messengers trigger the release of these hormones. Therefore, one should be aware of these limitation when interpreting the data in neuroendocrine challenge test. Also, it is possible that a change in production of a specific hormone will alter its response to a

5-HT1A agonist. Most 5-HT1A agonists have side effects to interact with other receptors, which can also increase the secretion of these hormones. Therefore, a

hormone response to a 5-HT1A agonist may not only reflect the function of 5-HT1A

receptor systems but also of other receptors. For example, buspirone is a 5-HT1A

agonist and a dopamine D2 antagonist. Since dopamine is a predominant controller of prolactin secretion, the increase in prolactin induced by buspirone may mainly represent

the D2 antagonist effect but not the activity of 5-HT1A receptors.

A number of approaches can be used to overcome these limitations of neuroendocrine challenge tests. First, it is necessary to test multiple hormone responses. Each hormone is differentially regulated in terms of their synthesis, their location and stimulation of their secretion by neurotransmitters. Therefore, the side

effect of 5-HT1A agonists may not influence all the hormones in a same manner. For example, in the present studies, repeated injections of paroxetine potentiated the ACTH and corticosterone responses to a high dose of 8-0H-DPAT. Since potentiation was not 153 observed in the oxytocin response, it is unlikely that the paroxetine-induced potentiation

of ACTH and corticosterone responses to 8-0H-DPAT is mediated by 5-HT1A receptors. Secondly, using more than one 5-HT1A agonist for the hormone challenge

test also helps to distinguish between hormone responses mediated by 5-HT1A receptors

and responses mediated by other receptors. Different 5-HT1A agonists may have different side effects which may influence the secretion of hormones. These side effects

of 5-HT1A agonists on the hormone responses may be different between 5-HT1A

agonists, while they share the same effects on 5-HT1A receptor-mediated hormone

responses. Thirdly, comparing the hormone responses to 5-HT1A agonists with the response to other receptor agonists, such as 5-HT2Anc agonists, can rule out the

possibility that changes in the hormone responses to 5-HT1A agonists are due to an alteration in the production of the hormone. For example, long-term injections of

fluoxetine decrease the hormone responses to 5-HT1A agonists but potentiate the hormone responses to a 5-HT2Aizc agonist (Li et al.1993a). suggesting that the decrease

in hormone responses to 5-HT1A agonists is not due to a reduction in the synthesis of these hormones.

An advantage of hormone challenge test is that the hormone responses to 5-HT1A

agonists amplify the changes in the 5-HT1A receptor systems. For example, activation

of 5-HT1A receptors on parvocellular cells of the PVN increases the secretion of CRF.

CRF further increase the secretion of ACTH from corticotrophs in the pituitary.

ACTH will further increase corticosterone release from the adrenal cortex. Along the

HPA axis, the signal of any change in the 5-HT1A receptors is enlarged in the each step. 154

This amplification is beneficial to monitor changes in 5-HT1A receptors.

As we hypothesized, desensitization of hypothalamic 5-HT1A receptors may play

a role in the therapeutic effects of SSRI's. It is possible that the changes in the 5-HT1A receptors occur prior to the improvement of clinical symptoms in patients receiving

SSRI's. Therefore, neuroendocrine challenge tests can proYide early information for physicians to monitor and predict the therapeutic effects of SSRI's.

Comparison of Desipramine with Fluoxetine in Neuroendocrine Challen1:e tests

In the present studies, we compared the effect of desipramine and fluoxetine on

the function of hypothalamic 5-HT1A receptors. Desipramine is a norepinephrine uptake inhibitor and a , while fluoxetine is a 5-HT uptake inhibitor and an atypical antidepressant (De Vane, 1994). The results of the present studies indicate that repeated injections of fluoxetine, but not desipramine, produce a desensitization of

hypothalamic 5-HT1A receptors. These results suggest that the desensitization of

hypothalamic 5-HT1A receptors is selectively induced by inhibition of 5-HT uptake but not by inhibition of norepinephrine uptake. Other studies agree with these observations.

For example, Lesch and co-investigators (1991; 199lc) observed that long-term treatment with fluoxetine decreases the ACTH response to ipsapirone in patients with obsessive-compulsive disorder, while a tricyclic antidepressant, , did not change the ACTH response to ipsapirone in depressed patients.

In contrast with the differential effect of fluoxetine and desipramine on the

hypothalamic 5-HT1A receptors, both fluoxetine and desipramine potentiated the

hormone responses to the 5-HT2 agonists DOI and MK-212 (Li et al.1993a). 155

Fluoxetine increased the density of the high affinity state 5-HT2A12c receptors in the hypothalamus (Li et al.1993a and our unpublished data). These observations are consistent with results from other studies (Li et al.1993a; Hulihan-Giblin et al.1994;

Cadogan et al.1993; Hrdina and Vu, 1993) and suggest that both 5-HT uptake inhibitors and tricyclic antidepressants increase the function of 5-HT 2A 12c receptors in the hypothalamus, although the mechanism of the supersensitivity of 5-HT2A; receptors may be different between the two classes of antidepressants. The fact that both 5-HT uptake inhibitors and tricyclic antidepressants have similar therapeutic effects on depression suggests that the sensitization of 5-HT2A12c receptors may play a role in therapeutic effects of the antidepressants in the treatment of depression.

Comparison of the Effects of Fluoxetine with Paroxetine on Hypothalamic 5- HT lA Receptors

To study whether the desensitization of hypothalamic 5-HT1A receptors, induced by repeated injections of fluoxetine, is due to blockade of 5-HT uptake sites, we compared the effects of fluoxetine with another 5-HT uptake inhibitor, paroxetine.

Paroxetine has a chemical structure and pharmacokinetic profile that is different from

fluoxetine. For example, fluoxetine has a long t112 and an active metabolite

(norfluoxetine) which has a similar potency for 5-HT uptake sites and a longer half-life than fluoxetine. Paroxetine has more than 10-fold higher affinity for 5-HT uptake sites, a shorter half-life and no active metabolites. Also, due to difference in chemical structure, paroxetine and fluoxetine may have different side effects. For example,

fluoxetine has about 20-fold higher affinity for 5-HT2A 12c receptors than paroxetine, 156 while paroxetine has a much higher affinity for muscarinic receptors than fluoxetine

(Stanford, 1996). On the other hand, both fluoxetine and paroxetine share similar effects in inhibiting 5-HT uptake sites. Therefore, if both fluoxetine and paroxetine

produce a desensitization of 5-HT1A receptors, then this desensitization most likely is mediated by the blockade of 5-HT uptake sites. As was shown in Chapter V and VI, both fluoxetine and paroxetine reduced the neuroendocrine responses to 8-0H-DPAT

and the level of Gi3 proteins in the hypothalamus, but did not alter the density of 5-HT1A receptors. These results suggest that the desensitization of hypothalamic 5-HT lA receptors is mediated by the blockade of 5-HT uptake sites. Furthermore, the

desensitization of hypothalamic 5-HT1A receptors is not due to receptor down­ regulation, but may partly be mediated by a reduction in the hypothalamic level of Gi3 proteins.

The effects of fluoxetine and paroxetine on the levels of Gi and G0 proteins are not similar. Overall, repeated injections of paroxetine produce a greater and earlier reduction in the hypothalamic levels of Gi proteins, especially the levels of Gil and Gi2 proteins, than that seen with fluoxetine. So far, there is no explanation for the

differential effects on the levels of Gi and G0 proteins between fluoxetine and

paroxetine. It is possible that paroxetine reduces the levels of Gi and G0 proteins not only because of blockade of 5-HT uptake sites, but also via stimulation of other receptors, such as muscarinic receptors, for which paroxetine has a higher affinity than

fluoxetine (Stanford, 1996). 5-HT1A agonists induce a faster desensitization of 5-HT1A receptors than 5-HT uptake inhibitors (De V ry, 1995). If this is the case for muscarinic 157 agonists and if paroxetine acts as an agonist, then the stimulation of muscarinic receptors may explain the earlier effects of paroxetine on the levels of some G proteins.

However, too little information is available on the regulation of the level of G proteins, and future studies are needed to clear this issue.

Fluoxetine and paroxetine are used to treat depressed patients. The efficacy of the antidepressants is similar. However, paroxetine appears to have an earlier onset of therapeutic effect on depression than fluoxetine (Lane et al.1995; Nemeroff, 1993;

Schone and Ludwig, 1993). This earlier onset of therapeutic effect is consistent with our observation that paroxetine induces an earlier reduction in the levels of G proteins.

On the other hand, the side effects induced by fluoxetine or paroxetine are somewhat different. Sedation and nausea are common with paroxetine, while anxiety, agitation and are common with fluoxetine. Since paroxetine does not induce anxiety and agitation, fewer patients will drop out from the treatment. Although the mechanism for the difference between paroxetine and fluoxetine is still unknown, it is possible that the earlier onset of therapeutic effect and the fewer side effects of paroxetine are due to its higher selectivity for 5-HT uptake sites.

Possible Mechanism for the Desensitization of Hypothalamic 5-HT tA Receptors

In the present studies, we observed that repeated injections of 5-HT uptake

inhibitors produce a delayed and gradual desensitization of hypothalamic 5-HT1A receptors as evidenced by a reduction in the neuroendocrine responses to 8-0H-DPAT.

The mechanism for the desensitization is still unknown. Most evidence has shown that

A 5-HT1 receptors are coupled to Gi or G0 proteins, which inhibit adenylyl cyclase, close 158 ca++ channels and open K+ channels. Therefore, any changes in the 5-HT1A receptors,

Gi proteins and adenylyl cyclase could alter the overall function of 5-HT1A receptors.

Few studies have investigated the regulation of hypothalamic 5-HT1A receptors.

Newman et al. (1992) observed that repeated injections of fluoxetine for 3 weeks reduce the inhibitory effect of 5-HT on forskolin-stimulated adenylyl cyclase in the hippocampus, suggesting that chronic fluoxetine-induced desensitization could be

mediated by changes at the receptor level, signal transduction system of 5-HT1A receptors, or the second messenger, adenylyl cyclase. It can be ruled out that a down­

regulation of the hypothalamic 5-HT1A receptors is involved in their desensitization,

because the density of 5-HT1A receptors in the hypothalamus and other brain regions was not altered by repeated injections of either fluoxetine or paroxetine. Also, a lack of change in the Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding suggests that

the desensitization of 5-HT1A receptors is not due to a decrease in the ability of 5-HT1A receptors to couple to their G proteins. However, it is still possible that the coupling

of 5-HT1A receptors to the G proteins that link to the release of hormones is decreased

in living cells. There is differential affinity of 5-HT1A receptor coupling to G proteins

(Gi3 > Gi1 > Gi2 > G0 proteins) (Mulheron et al.1994; Raymond et al.1993).

Therefore, a reduction in the level of Gi3 proteins could alter the proportion of G

proteins that are coupled to 5-HT1A receptors. This change can not be determined by

Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding, while it can result in a

reduction of the function of 5-HT1A receptors. Few studies have reported on the effect

of 5-HT uptake inhibitors on levels of G proteins that are coupled to 5-HT1A receptors. 159

Results of the present studies suggest that the decrease in the level of Gi3 proteins may

play a role in the desensitization of the hypothalamic 5-HT1A receptors. However, since it is still unknown whether Gi3 proteins mediate 5-HT1A receptor-induced secretions of

ACTH, corticosterone and oxytocin, it seems too early to conclude that the reduction

in the level of Gi3 proteins mediates the desensitization of 5-HT 1A receptors.

Another possible mechanism for the desensitization of the hypothalamic 5-HT1A receptors is their phosphorylation. Supersensitivity of 5-HT2A receptors induced by repeated injections of 5-HT uptake inhibitors could be a cause of desensitization of 5-

HT1A receptors. Increasing of the activity of 5-HT2A receptors will stimulate protein kinase C, which in tum can phosphorylate 5-HT1A receptors. 5-HT1A receptors contain at least three potential sites for phosphorylation by protein kinase C (Raymond et al.1992). Phosphorylation of 5-HT1A receptors induces a desensitization of the 5-HT1A receptors (Harrington et al.1994; Nebigil et al.1995; Raymond and Olsen, 1994; van

Huizen et al.1993; Raymond, 1991). It has been reported that 5-HT1A agonist-induced desensitization of 5-HT1A receptors in HeLa cells can be mimicked by a protein kinase

C activator and by forskolin, which activates adenyly 1 cyclase and consequently

activates protein kinase A. The desensitization of 5-HT1A receptors induced by 8-0H­

DPAT can be blocked by inhibitors of protein kinase C, suggesting that protein kinase

C can be involved in the desensitization of 5-HT1A receptors (Harrington et al.1994).

This result is consistent with some studies (Raymond and Olsen, 1994; Raymond,

1991), while yet other studies suggest that the phosphorylation of 5-HT1A receptors might be mediated by another G-protein-activated protein kinase, but not by protein 160 kinase C (Nebigil et al.1995; van Huizen et al.1993). All these data suggest that 5-

HT IA receptors can be phosphorylated and consequently be desensitized, although it is

still unknown whether phosphorylation of 5-HT1A receptors alters their binding of

agonists or their coupling to G proteins. However, it should be noted that most of these studies were performed in cells expressing cloned 5-HT IA receptors. To date, no data are available regarding the effect of 5-HT uptake inhibitors on the phosphorylation

of 5-HT1A receptors.

G proteins function as a linkage between receptors and second messengers.

Activation of receptors by agonists induces an exchange of GDP by GTP on the a subunits of G proteins, resulting in a dissociation of the ex subunits from the B'Y complex of the G proteins and the receptors. The a subunits of G proteins then activate or inhibit second messenger systems. On the other hand, the dissociation of a subunits from their B'Y complex activates the GTPase activity of a subunits of G proteins and hydrolyzes the GTP to GDP, resulting in a reduced ability of G" proteins to interact with adenylyl cyclase and recombination of a subunits with fi)' subunits.

Therefore, if the GTPase activity were increased, the a subunits of G proteins would be inactivated faster and reduce the signal transduction of receptors. An in vitro study has recently reported that three regulators of G protein signaling (RGS), GAIP (G

Alpha Interacting Protein), RGS4 and RGSlO, activate the GTPase activity of a

subunits of Gi proteins, especially of Gi3 proteins (Berman et al.1996; Watson et al.1996; Koelle and Horvitz, 1996; Hunt et al.1996; De Vries et al.1995). RGS4 has been found in the rat brain (Watson et al. 1996). If the effect of RGS proteins on the 161

GTPase activity also occurs in vivo, it can be hypothesized that desensitization of

hypothalamic 5-HT1A receptors, induced by repeated injections of 5-HT uptake inhibitors, may be due to an increase of RGS4, GRSlO or GAIP in the hypothalamus.

The increase of RGS4 and/or GAIP results in an increase in the GTPase activity of a subunits of Gi proteins, and consequently, increases in hydrolysis of GTP and inactivation of Gai proteins. This rapid inactivation of a subunits of Gi proteins will decrease the function of the second messenger related to 5 -HT IA receptors. This hypothesis can account for the results of the present studies, i.e. that repeated injections

of 5-HT uptake inhibitors decrease the function of the hypothalamic 5-HT1A receptors

without changing the density of 5-HT1A receptors and the coupling of 5-HT1A receptors to their G proteins. However, it is still unknown whether repeated exposure to 5-HT uptake inhibitors alters the levels of RGS4, RGSlO and/or GAJP in the hypothalamus.

This hypothesis is currently being investigated.

It is also possible that the desensitization of 5-HT1A receptors results from an

interaction between 5-HT1A receptors and other receptors. 5-HT1A receptors share Gi proteins with several other receptors, such as D2 dopamine receptors, GABAs receptors,

M2 muscarinic receptors and µ opiate receptors (Odagaki and Fuxe, 1995; Innis and

Aghajanian, 1987; Innis et al.1988). However, no evidence, so far, has shown that 5-

HT uptake inhibitors influence these receptors.

Beside the mechanisms discussed above, other regulations of the G proteins and

second messenger systems can also induce the desensitization of 5-HT1A receptors. For

example, alterations in the linkage between G proteins that are coupled to 5-HT1A 162 receptors and adenylyl cyclase may change the function of 5-HT1A receptors. Since Gi proteins are negatively coupled to adenylyl cyclase, increases in the concentration of

cAMP may eventually decrease the function of 5-HT1A receptors. So far, no study on

the effect of 5-HT uptake inhibitors on palmitoylation of 5-HT1A receptors has been reported.

Si1mificance of the Present Studies

Results of the present studies indicate that repeated injections of 5-HT uptake

inhibitors produce a delayed and gradual desensitization of the hypothalamic 5-HT1A

receptors as examined by reduction in the hormone responses to S-HT1A agonists. This result is consistent with report by Lesch et al. (1991c), who found that long-term administration of fluoxetine reduces the ACTH response to ipsa.pirone in patients with obsessive compulsive disorder. These results support the hypothesis that repeated

injections of 5-HT uptake inhibitors induce an adaptive change in postsynaptic 5-HT1A receptors, which may play a role in the therapeutic effects of 5-HT uptake inhibitors.

Most 5-HT uptake inhibitors produce a desensitization of 5-HT1A receptors, while most tricyclic antidepressants desensitize B adrenergic receptors (Bourin and

Baker, 1996). It is still unknown what is the significance of the desensitization of

hypothalamic 5-HT1A receptors in terms of their therapeutic effects. One possibility is

that the desensitization of hypothalamic 5-HT1A receptors could be related to the treatment of a subgroup of depressed patients, whose symptoms are due to impaired serotonergic neurotransmission. According to the "amine hypothesis" , depression can be initiated by impaired adrenergic and/or serotonergic systems. It is not surprising 163 that a subgroup of depressed patients who suffer from impaired serotonergic systems will be more responsive to 5-HT uptake inhibitors than those who have impaired adrenergic systems (Leonard, 1993; Asberg and Martensson, 1993). The

desensitization of hypothalamic 5-HT1A receptors may be a benefit for this group of patients. In fact, fluoxetine has been used to treat melancholia, which does not respond to tricyclic antidepressants (Asberg and Martensson, 1993). File and her colleagues

(1996; 1994) reported that 5-HT1A autoreceptors in the median raphe are involved in

the anxiolytic effect of 5-HT1A agonists, while stimulation of postsynaptic 5-HT1A receptors in the dorsal hippocampus produces an anxiogenic effect. So far, no clinical evidence supports their observation. However, if these observations are also correct

in human, then the desensitization of postsynaptic 5-HT1A receptors by 5-HT uptake inhibitors may be related to the antianxiety effects of SSRI's.

Although SSRI' s and tricyclic antidepressants have similar efficacy for depression, SSRI's are also used to treat other affective disorders, such as obsessive compulsive disorder, premenstrual syndrome, and eating disorders. Tricyclic antidepressants are ineffective for these disorders. The desensit1zation of hypothalamic

5-HT1A receptors may play a role in these specific applications. Recent evidence has suggested that SSRI's can be used to treat schizophrenia (Breier, 1995; Costall and

Naylor, 1992). Since D2 dopamine receptors share Gi proteins with 5-HT1A receptors, the decrease in the Gi proteins induced by SSRI' s may be beneficial for the treatment of schizophrenia.

Another possible beneficial effect of the desensitization of 5-HT IA receptors is 164 the development of tolerance for the side effects of SSRI's (De Vry, 1995). In general, both tricyclic antidepressants and SSRI's have similar therapeutic effects for depression

(Owens, 1994; Harrison, 1994; Blier and de Montigny, 1994; Montgomery, 1994;

Wong et al.1995). However, tricyclic antidepressants have severe side effects, such as tachycardia, hypertension and impairment of motor and cognitive functions. An overdose of tricyclic antidepressants can result in cardiovascular disorders and even death (Blier and de Montigny, 1994; Montgomery, 1994; Wong et al.1995; Leonard,

1993). On the other hand, SSRI's have much fewer severe side effects and are better tolerated. This benefit makes SSRI's have a lower dropout rate than tricyclic antidepressants (Lane et al.1995; Gram, 1994). One could expect that the different

effect of SSRI's and tricyclic antidepressants on hypothalamic 5-HT1A receptors may be involved in the differential side effects of these two classes of antidepressants (De Vry,

1995).

Although both 5-HT1A agonists and 5-HT uptake inhibitors induce a desensitization of 5-HT1A receptors, neither group of drugs decreases the density of 5-

HT1A receptors (De Vry, 1995; Chen et al.1995; Hensler et al.1991). This suggests that the regulation of 5-HT1A receptor systems is usually not at the receptor level.

Tricyclic antidepressants increase G protein activation of adenylyl cyclase (Ozawa and

Rasenick, 1989; Chen and Rasenick, 1995a; Ozawa et al. 1994), while repeated

injections of 5-HT uptake inhibitors reduce the levels of Gi and G0 proteins in a region­ specific manner, suggesting that G proteins and signal transduction systems may be the target of antidepressants. Therefore, understanding the role of signal transduction 165 systems in the treatment of affective disorders will be beneficial for the development

of new antidepressants (Broekkamp et al .1995).

Limitation of the Present Studies

Several limitations should be noted when interpreting the data in the present

studies. First, the present studies were performed with normal male rats, in which

neurotransmission may be different from that in depressed patients. Therefore, it is

possible that desensitization of hypothalamic 5-HT1A receptors does not occur in depressed patients. However, Lesch et al (1991c) observed that long-term treatment of obsessive compulsive disorder patients with fluoxetine reduced the ACTH response to ipsapirone. Furthermore, the time-course of the desensitization of hypothalamic 5-

HT IA receptors may be different from the time-course of therapeutic effect in humans, especially since the pharmacokinetics of 5-HT uptake inhibitors in rats are different from those in humans. For example, the dose of fluoxetine used in rats was much higher than the dose used in humans, but the half-life of f111oxetine in rats is much shorter than the half-life in humans (1 day vs. 5 hours) (1990).

In the present studies, hormone responses to 5-HTM agonists were used to examine the function of 5-HT IA receptors. The hormone challenge test has been used

in both experimental animals and humans to study the function of 5-HT1A receptors.

However, these hormone responses reflect mainly 5-HT1A receptors which control neurons in the parvocellular and magnocellular regions of the paraventricular nucleus

of the hypothalamus. It is still unknown whether the changes in 5-HT1A receptors in the neuroendocrine cells can reflect changes in other neurons in the hypothalamus or 166 even in other brain regions. So far, only a few studies have reported on the effects of

repeated exposure to 5-HT uptake inhibitors on the function of postsynaptic 5-HT1A receptors (Newman et al.1992; Hensler et al.1991). Since our data suggest that the effects of 5-HT uptake inhibitors on the levels of G proteins are region-specific, it is possible that the effects of 5-HT uptake inhibitors on the 5-HT1A receptor systems are also region-specific.

Signal transduction of 5-HT1A receptors was evaluated in these studies by

examining the levels of Gi and G0 proteins. The limitations of thjs approach are, first,

that little is known about the specific Ga proteins that are involved in 5-HT1A receptor­ induced increases in the secretion of ACTH and oxytocin. Secondly, the reduction of

the levels of G proteins is not necessarily related to the function of 5-HT1A receptors, since several receptors are coupled to the same G proteins. Therefore, better methods

should be used to determine the signal transduction of 5-HT1A receptors, such as agonist-stimulated [35S]-GTP')"S [35S-guanosine-5 '-0-(thiotriphosphate)] binding and agonist-dependent photolabeling of G protein a subunits with [32P]GTP azidoanilide.

Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding was used to examine

the coupling of 5-HT1A receptors to the G proteins in the present studies. Gpp(NH)p is a non-hydrolyzable GTP analog, which exchanges with GDP a.nd binds to a subunits of G proteins when agonists bind to the receptors. The binding of Gpp(NH)p to the a subunits of G proteins will dissociate the G proteins from the receptors and tum the receptors from their high affinity state to the low affinity state. Therefore, in the

presence of Gpp(NH)p, agonist-binding will be inhibited. In the case of 5-HT1A 167

receptors, Gpp(NH)p will inhibit 3H-8-0H-DPAT binding. The percent of the

inhibition was used to represent the degree of 5-HT1A receptors which are coupled to

G proteins. However, it should be noted that the assay was performed in vitro in a

frozen brain slice and only 3H-8-0H-DPATwas incubated with the brain sections. This

is different from the normal environment surrounding 5-HT IA receptors in living cells.

Therefore, the degree of Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding can

only reflect the steady-state coupling of 5-HT1A receptors to G proteins. In other

words, Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding is more dependent on

the ability of 5-HT1A receptors to couple to their G proteins than on the condition of these G proteins. For example, the degree of Gpp(NH)p-induced inhibition of 3H-8-

0H-DPAT binding may not reflect changes in the levels of Gprote:ins, but may indicate

the ability of 5-HT1A receptors to couple to their G proteins. Therefore, a lack of change in the percentage of Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT binding

suggests that fluoxetine does not alter the ability of 5-HT1A receptors to couple to their

G proteins.

Conclusions

The present results suggest that prolonged blockade of 5-HT uptake sites produces a delayed and gradual desensitization of the hypothalamic 5-HT IA receptors.

This desensitization is not due to a decrease in the density of S-HT1A receptors in the

hypothalamus because no change in the density of 5-HT1A receptors has been detected after repeated injections of either fluoxetine or paroxetine. Farthermore, a lack of changes in the Gpp(NH)p-induced inhibition of 3H-8-0H-DPAT bjnding suggests that 168 the coupling of 5-HT lA receptors to G proteins is not altered by repeated injections of the 5-HT uptake inhibitors. However, it is possible that blockade of 5-HT uptake sites

changes the proportion of different types of G proteins that are coupled to 5-HT1A receptors due to a reduction in the levels of one or two of the G proteins, resulting in

changes in the function of 5-HT1A receptors. It has been shown that one receptor can couple to several types of G proteins with various affinities (Mons and Cooper, 1995;

Rudolph et al.1996; Yung et al.1995; Kozasa and Gilman, 1995; Chan et al.1995).

The differential coupling to G proteins can alter the efficacy of the receptor in terms of its function. This could be due to a different linkage between G proteins and second messengers. For example, Gz proteins produce more potent inhibition of type V

adenylyl cyclase than Gi 1 proteins (Kozasa and Gilman, 1995). Another possible reason for the alteration of efficacy is that the rate of dissociation of G proteins and receptors is different between the G protein subtypes (Kozasa and Gilman, 1995). In the present studies, we demonstrated that blockade of 5-HT uptake sites reduces the hypothalamic

levels of Gil and Gi3 proteins, but not Gi2 proteins. Since Gi1 and Gi3 proteins have a

higher affinity for 5-HT1A receptors than Gi2 and G0 proteins (Raymond et al.1993), one can expect that Gi 1 and Gi3 proteins are the majority of G pmteins coupled to the 5-

HT 1A receptors in the normal condition. When the levels of Gi1 and Gi3 proteins are

reduced, the ratio of Gi1, Gi2, Gi3 and G0 proteins coupled to j-HT1A receptors may be changed. This would result in an alteration in the linkage 10 second messengers or a

reduction in the efficacy, and subsequently a decrease in the function of 5-HT1A receptors. The results in the present studies suggest that a reduction in the 169 hypothalamic level of Gi3 proteins may be involved in the desensitization of 5-HT1A receptors, because there is a similarity of the time courses in reducing the hypothalamic level of Gi3 proteins and the hormone responses to 5-HT 11~. agonists after repeated injections of fluoxetine and paroxetine.

In conclusion, the results of the present studies support our hypothesis that repeated injections of 5-HT uptake inhibitors produce a delayed and gradual desensitization of hypothalamic 5-HT lA receptors. The mechanism of the desensitization

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The author, Qian Li, was born on January 1, 1955, in Shanghai, People's Republic of China. She received her Master of Science degree in Biochemistry at Chongqing

University of Medical Science on January of 1985. During the following five years,

Ms Li worked as research associate and then Assistant professor in the Department of

Biochemistry at Changqing University of Medical Science in China. In October, 1990,

Ms Li came to the United States and worked at Dr. Louis D. Van de Kar's laboratory as a research associate in the Department of Pharmacology at Loyola University

Chicago. In the summer of 1992, Ms. Li was accepted into the graduate program in the Department of Pharmacology at Loyola University Chicago. Ms. Li rejoined Dr.

Van de Kar's laboratory and developed her dissertation project under the guidance of

Dr. Louis D. Van de Kar.

Ms. Li was awarded a 2 year predoctoral fellowship from the Pharmaceutical

Research and Manufacturers of America foundation association in 1995. She also received a Young Investigator award from the rnternational Serotonin Meeting in 1994.

Ms. Li is a member of the Society of Neuroscience and the American Society for

Pharmacology and Experimental Therapeutics.

Ms. Li has accepted a position as a postdoctoral fellow in the laboratory of Dr.

Dennis L. Murphy in the Laboratory of Clinical Science at the National Institute of

Mental Health in Bethesda, Maryland.

202 DISSERTATION APPROVAL SHEET

The dissertation submitted by Qian Li has been read and approved by following committee:

Louis D. Van de Kar, Ph.D. (Dissertation advisor) Professor Department of Pharmacology Loyola University Chicago

George Battaglia, Ph.D. Associate Professor Department of Pharmacology Loyola University Chicago

Thackery S. Gray, Ph.D. Professor Department of Cell Biology, Neurobiology and Anatomy Loyola University Chicago

Israel Hanin, Ph.D. Professor and Chairman Department of Pharmacology Loyola University Chicago

Mary D. Manteuffel, Ph.D. Professor Department of Molecular and cellular Biochemistry Loyola University Chicago

Nancy Muma, Ph.D. Associate Professor Department of Pharmacology Loyola University Chicago

The final copies have been examine by the director of the dissertation and the signature which appears below verifies the fact that any necessary changes have been incorporated and that the dissertation is now given final approval by the committee with reference to content and form.

The dissertation is, therefore, accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy. /'/. / f / I l-/ ' / . '-\_, , I Date Director's Signature